Regenerative human type I collagen, medical material and preparation method thereof

Abstract

The invention discloses a regenerative human type I collagen, a medical material and a preparation method thereof, wherein the preparation method of the regenerative human type I collagen comprises isolating and proliferating human skin cells or mesenchymal stem cells in vitro; then the cells are inoculated into a proliferation medium for static culture, and then dynamically cultured in a secretagogue protein medium; wherein the secretagogue protein medium comprises a basal medium and factors; the factors include magnesium ascorbyl phosphate, glucocorticoids, and insulin transferrin. The above preparation method is controllable in operation, can be beneficial to eliminate batch differences, and ensure uniform and stable mass production; after static proliferation culture, the cell culture process uses a secretagogue protein medium containing specific factors for dynamic culture, and the stimulating effect of the factors on the cells can be utilized to better promote amino acid synthesis and secretion of collagen, thereby improving the yield of type I collagen; and the prepared collagen is derived from humans, which can eliminate animal virus hazards.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a regenerated human type I collagen, a medical material and a preparation method thereof. Background Art

[0002] Collagen, also known as collagen, is the most abundant protein in mammals, accounting for about 25-30% of the total protein in the body. It is widely present in the skin, bones, cartilage, teeth, tendons, ligaments and blood vessels of animals. It is an important structural protein of connective tissue, playing a role in stabilizing tissues and organs and maintaining their structural integrity. In addition, collagen can provide the necessary nutrients for the skin layer containing collagen, enhance the activity of collagen in the skin, maintain the moisture of the stratum corneum and the integrity of the fiber structure, improve the living environment of skin cells and promote the metabolism of skin tissue, increase circulation, and achieve the purpose of moisturizing the skin, delaying aging, beautifying, removing wrinkles, and nourishing hair.

[0003] The traditional method of producing collagen is to use acid, alkali or enzyme to treat animal tissues (pig skin, cow skin, donkey skin, fish skin / scales, etc.) to extract collagen. Although these methods are low-cost, they are derived from animals and have the risk of animal immunogenicity and viral residues. After implantation into the human body, they may cause foreign rejection reactions and the risk of introducing animal-derived viruses into the human body, limiting their application as biomaterials in medical devices and other fields; and the mass production uniformity and stability of these methods are difficult to guarantee, because the uniformity of the epidermal thickness of animals of different ages in different regions varies greatly, which will affect the batch-to-batch stability and consistency of the product during subsequent processing and application. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a regenerated human type I collagen, a medical material and a preparation method thereof.

[0005] In a first aspect of the present invention, a method for preparing regenerated human type I collagen is provided, which comprises the following steps:

[0006] S11. Isolating and proliferating human skin cells or mesenchymal stem cells in vitro;

[0007] S12, inoculating the cells into a proliferation medium for static culture; and then culturing them dynamically in a secretory protein medium; the secretory protein medium comprises a basal medium and factors; the factors in the secretory protein medium comprise 20ug / mL to 60ug / mL of magnesium ascorbyl phosphate, 0.02ug / mL to 0.05ug / mL of glucocorticoids, and 0.02ug / mL to 0.06ug / mL of insulin transferrin.

[0008] According to the preparation method of the embodiment of the present invention, the regeneration of human type I collagen has at least the following beneficial effects: the type I collagen prepared by the method is derived from humans, which can effectively eliminate the risks of immunogenicity and viral residues in animal-derived collagen; the specific operation of the preparation method is controllable, which is conducive to eliminating batch differences and ensuring mass production uniformity and stability. In the specific preparation process, the cells obtained by in vitro separation and proliferation culture are first inoculated into a proliferation culture medium for static proliferation culture, and then replaced with a secretory protein culture medium containing specific factors for dynamic culture. On the one hand, the factor glucocorticoid in the secretory protein culture medium can effectively inhibit the proliferation and division of cells, and the factor ascorbic acid phosphate magnesium salt and insulin transferrin can effectively promote the secretion of collagen by cells. The stimulation of these three factors on cells can better promote amino acid synthesis and secretion of collagen, thereby improving the yield of type I collagen.

[0009] In step S11, skin cells or mesenchymal stem cells derived from humans may be separated and cultured in vitro by tissue engineering to serve as raw materials for the subsequent production of sheet tissues.

[0010] In step S12, the cells can be inoculated into a tissue culture vessel treated with adherent TC for culture, and the tissue culture vessel can have 1 to 50 layers, for example, 1 layer, 3 layers, 5 layers, 8 layers, 10 layers, 15 layers, 20 layers, 22 layers, 25 layers, 28 layers, 30 layers, 35 layers, 40 layers, 46 layers, 50 layers, etc. The tissue culture vessel is designed as a rectangular container, a cube container, a cylindrical container, or a container of other shapes.

[0011] In some embodiments of the present invention, in step S12, the initial seeding density of cells inoculated into the proliferation medium is 6000 to 20000 cells / cm 2 Furthermore, the initial cell seeding density can be 8000 to 16000 cells / cm 2 10,000 to 20,000 cells / cm 2 or 10,000 to 15,000 cells / cm 2 .

[0012] In some embodiments of the present invention, in step S12, the static culture time is 2 to 4 days, for example, 2 days, 2.5 days, 3 days or 4 days.

[0013] In some embodiments of the present invention, the proliferation culture medium may be a proliferation-promoting culture medium kit. When the cell fusion degree is observed under a microscope to reach more than 95%, the cell fusion degree may be more than 95%.

[0014] Cell growth factors that promote cell proliferation are given to the proliferation medium. During the static culture process in the proliferation medium, the cells proliferate and divide rapidly. After the static culture is completed, the culture medium is replaced with a secretory protein culture medium, through which cell growth factors that effectively secrete collagen are given.

[0015] In some embodiments of the present invention, the amount of magnesium ascorbyl phosphate in the secretory protein culture medium may be 25ug / mL to 50ug / mL, 30ug / mL to 40ug / mL or 40ug / mL to 60ug / mL; the amount of glucocorticoid may be 0.03ug / mL to 0.05ug / mL or 0.02ug / mL to 0.04ug / mL; the amount of insulin transferrin may be 0.03ug / mL to 0.05ug / mL or 0.04ug / mL to 0.06ug / mL.

[0016] In some embodiments of the present invention, the factors in the secretagogue protein culture medium further comprise: 30 mg / mL to 70 mg / mL of glycine, alanine, proline, lysine, hydroxyproline and methionine in a mass ratio of 1:1:1:1:1:1, and / or, 2 wt.% to 10 wt.% of fetal bovine serum or a serum substitute.

[0017] In some embodiments of the present invention, in the secretagogue protein culture medium, the amount of glycine, alanine, proline, lysine, hydroxyproline and methionine in a mass ratio of 1:1:1:1:1:1 may be 35 mg / mL to 50 mg / mL, 40 mg / mL to 60 mg / mL or 50 mg / mL to 70 mg / mL; the amount of fetal bovine serum or serum substitute may be 4wt.% to 8wt.%, 3wt.% to 7wt.% or 5wt.% to 10wt.%.

[0018] In some embodiments of the invention, the factors in the secretagogue protein culture medium include 25 ug / mL of ascorbyl phosphate magnesium salt, 36 mg / mL of glycine, alanine, proline, lysine, hydroxyproline and methionine in a mass ratio of 1:1:1:1:1:1, 0.025 ug / mL of glucocorticoids, 0.025 ug / mL of insulin transferrin, and 3 wt.% of fetal bovine serum or serum substitute.

[0019] In some embodiments of the present invention, in step S12, the basal culture medium is selected from DMEM culture medium and / or MEM culture medium; and / or the glucocorticoid is selected from at least one of betamethasone, methylprednisolone, dexamethasone, and beclomethasone.

[0020] After switching to secretory protein culture medium, dynamic culture can be carried out by starting the culture medium circulation system. At this time, the cells no longer proliferate and divide rapidly, but basically maintain the existing cell amount. Under the stimulation of the secretory culture medium and the circulating dynamic culture medium, the cells begin to secrete collagen in large quantities, and show a superimposed secretion effect, thereby obtaining collagen-rich tissue.

[0021] In some embodiments of the present invention, in step S12, the dynamic culture is a cyclic dynamic culture, including: replacing the culture medium every 2 to 7 days, and controlling the circulation of the culture medium from the 2nd to 7th day of the dynamic culture to stimulate the circulating culture medium. Further, the time interval for replacing the culture medium can be controlled to be 3 to 6 days.

[0022] In some embodiments of the present invention, the circulation flow process of the culture medium is controlled, and the flow rate of the first liquid change can be controlled to be 5rpm~15rpm, the flow rate of the second liquid change can be controlled to be 15rpm~30rpm, and the flow rate of the third liquid change can be controlled to be 20rpm~40rpm, so as to promote the secretion of collagen and the formation of tissue.

[0023] In some embodiments of the present invention, the circulation flow process of the culture medium is controlled, and the flow rate of the first liquid change can be controlled to be 7 rpm, the flow rate of the second liquid change can be controlled to be 20 rpm, and the flow rate of the third liquid change can be controlled to be 36 rpm.

[0024] In some embodiments of the present invention, the total culture duration of the static culture and the dynamic culture is 13 to 28 days. Further, the total culture duration can be controlled to be 15 to 25 days or 20 to 25 days, for example, 15 days, 18 days, 20 days, 22 days or 25 days.

[0025] In some embodiments of the present invention, the preparation method further comprises: S13, collecting collagen.

[0026] In some embodiments of the present invention, in step S13, collagen is collected by in situ decellularization, specifically, the eluent is first used for in situ circulation perfusion for 4 to 8 hours, and then the PBS buffer solution is used for circulation perfusion 2 to 5 times, each time for 10 to 30 minutes.

[0027] In some embodiments of the present invention, the eluent used in the in situ decellularization treatment includes: sodium dodecyl sulfate (SDS), ethylenediaminetetraacetic acid (EDTA), sodium chloride (NaCl). Further, the eluent may include: 1.8mM SDS, 1M NaCl, 25mM EDTA.

[0028] The second aspect of the present invention provides a regenerated human type I collagen, which is prepared by any of the aforementioned methods for preparing regenerated human type I collagen.

[0029] The regenerated human type I collagen is derived from humans and is real human collagen. It is different from the recombinant collagen on the market (such as humanized, humanoid or recombinant human collagen). The regenerated human type I collagen is a high kDa collagen with a full chain length and a complete triple helix structure (composed of two α1 and one α2 chains). It can form a reticular fiber structure and has good repair properties. Compared with recombinant collagen, it has the advantages of high molecular weight, full range of amino acids, and true natural triple helix structure. Its spatial reticular structure can have a longer-lasting and more efficient repair effect than recombinant collagen. In addition, since the regenerated human type I collagen is derived from humans, it can effectively eliminate the risks of immunogenicity and viral residues in animal-derived collagen.

[0030] In a third aspect of the present invention, a medical material is proposed, comprising poly (L-lactic acid) microspheres and a functional coating layer loaded on the surface of the poly (L-lactic acid) microspheres, wherein the functional coating layer comprises collagen, and the collagen is any one of the aforementioned regenerated human type I collagen.

[0031] The medical material can be used as an implantable repair material and can be used for subcutaneous injection in the human body, such as subcutaneous injection in the face. The medical material is loaded with a functional coating layer containing regenerated human type I collagen on the surface of the L-polylactic acid microspheres, wherein the L-polylactic acid microspheres are biodegradable polymer materials, which have the characteristics of being non-toxic, non-irritating, biodegradable and absorbable, high in strength, good in plasticity, and easy to process and shape. After being injected into the body, they can be decomposed into L-lactic acid in the body, which is the same as the natural lactic acid in the human body. It can effectively stimulate the vitality of skin fibroblasts, stimulate the body's own collagen regeneration to fill the sunken parts, and improve facial wrinkles and skin quality, which can achieve volume contour changes and anti-aging all-round facial rejuvenation; and the L-lactic acid microspheres injected into the human body will show therapeutic effects after a few weeks or months, and continue to act in human tissues for up to three years. Regenerative human type I collagen comes from humans, which can effectively eliminate the hidden dangers of animal viruses and the risk of rejection from foreign sources. It also has good long-term repair properties. After being injected or implanted into the human body as a repair filling material, it can directly play a role and make wrinkles disappear. The compounding of L-polylactic acid microspheres and regenerative human type I collagen can maximize the efficacy of both, so that the medical materials of the product have efficient and long-term repair effects. Specifically, the fibroblast-stimulating effect of lactic acid after the decomposition of L-polylactic acid microspheres is combined with the induction and repair effect of regenerative human type I collagen, which can stimulate the vitality of skin fibroblasts and stimulate the regeneration of the body's own collagen. At the same time, combined with the regenerative human type I collagen contained in the medical material, it can effectively fill the sunken parts and improve facial wrinkles and skin quality, fully realizing the all-round facial youthfulness of volume contour changes and anti-aging; and, after the material is filled and implanted into the human body, it combines well with human tissues, reduces foreign body rejection reactions, and meets clinical safety requirements. This medical material has broad application prospects in clinical skin trauma repair, surgical patches, cartilage repair, medical beauty injections, cosmetics and other industries.

[0032] In some embodiments of the present invention, the functional coating layer is a collagen layer or a gel layer containing collagen.

[0033] In some embodiments of the present invention, the gel layer is any one of a sodium alginate gel layer, a sodium hyaluronate gel layer, and a chitosan gel layer.

[0034] In some embodiments of the present invention, the particle size of the L-polylactic acid microspheres is 30 μm to 35 μm.

[0035] In some embodiments of the present invention, the medical material further comprises an adsorption-promoting layer; the adsorption-promoting layer is disposed between the L-polylactic acid microspheres and the functional coating layer. The provision of the adsorption-promoting layer can improve the compositeness of the functional coating layer and the L-polylactic acid microspheres, reduce the risk of separation after implantation in the human body, and improve the long-term implantation repair effect.

[0036] In some embodiments of the present invention, the adsorption promoting layer is any one of a polyethyleneimine layer, a polydopamine layer, and a calcium phosphate layer.

[0037] A fourth aspect of the present invention provides a method for preparing the aforementioned medical material, comprising the following steps:

[0038] S21, preparing a coating slurry using a functional coating layer raw material including collagen; wherein the collagen is the aforementioned regenerated human type I collagen;

[0039] S22, mixing the L-polylactic acid microsphere powder with the coating slurry.

[0040] The coating slurry prepared in step S21 can be an aqueous solution or gel containing collagen; further, the gel containing collagen can be sodium alginate gel containing collagen, sodium hyaluronate gel containing collagen, or chitosan gel containing collagen.

[0041] In some embodiments of the present invention, step S21 includes mixing collagen and solvent water to prepare a collagen aqueous solution as a coating slurry. Specifically, the collagen and solvent water can be mixed and stirred until dissolved, and the coating slurry can be obtained after defoaming; wherein, the mixing and stirring can be carried out by magnetic stirring, and the stirring time can be controlled at 30min to 90min, for example, 30min, 45min, 60min or 90min; the defoaming can be carried out by standing defoaming, and the standing time can be controlled at 20min to 40min, for example, 20min, 25min, 30min or 40min; the amount of collagen in the coating slurry can be controlled to 0.0005g / mL to 0.0015g / mL, for example, 0.0005g / mL, 0.001g / mL, 0.0012g / mL or 0.0015g / mL.

[0042] In some embodiments of the present invention, step S21 includes preparing a collagen-containing gel as a coating slurry using a functional coating layer raw material including collagen and a gel precursor, wherein the gel precursor may be sodium alginate, sodium hyaluronate or chitosan.

[0043] In some embodiments of the present invention, step S21 includes preparing a sodium alginate gel containing collagen using a functional coating layer raw material including collagen and sodium alginate as a coating slurry.

[0044] Specifically, collagen and sodium alginate can be dissolved and dispersed in water, fully swollen, and then mannitol is added to adjust the osmotic pressure to obtain a coating slurry. The amount of collagen in the coating slurry can be controlled at 3wt.% to 8wt.%, for example, 3wt.%, 5wt.%, 6wt.% or 8wt.%; the amount of sodium alginate can be controlled at 0.5wt.% to 3wt.%, for example, 0.5wt.%, 1wt.%, 1.5wt.%, 2wt.% or 3wt.%. In addition, the osmotic pressure of the coating slurry can be controlled to 250mosm / L to 350mosm / L (such as 250mosm / L, 300mosm / L or 340mosm / L), or the amount of mannitol in the coating slurry can be controlled to 2wt.% to 6wt.% (such as 2wt.%, 3wt.%, 4wt.% or 6wt.%).

[0045] In some embodiments of the present invention, step S21 includes preparing a sodium hyaluronate gel containing collagen using a functional coating layer raw material including collagen and sodium hyaluronate as a coating slurry.

[0046] Specifically, collagen can be dissolved in water to obtain a collagen aqueous solution; sodium hyaluronate is dissolved in a sodium hydroxide solution, divinyl sulfone is added, the temperature is raised to 50°C to 80°C and stirred for reaction to obtain a cross-linked sodium hyaluronate gel, which is then placed in a hydrochloric acid solution and stirred for reaction, the hydrochloric acid solution is then filtered off, a phosphate buffer solution is added to dissolve, and finally the gel is evenly mixed with the collagen aqueous solution to obtain a coating slurry.

[0047] Among them, sodium hyaluronate with a weight average molecular weight of about 160,000 can be used, and the concentration of sodium hydroxide can be 1.5 mol / L to 2.5 mol / L, such as 1.5 mol / L, 1.8 mol / L, 2 mol / L or 2.5 mol / L; sodium hyaluronate is dissolved in a sodium hydroxide solution, and the mass concentration of sodium hyaluronate in the solution can be controlled to be 15 wt.% to 25 wt.%, such as 15 wt.%, 20 wt.%, 22 wt.% or 25 wt.%; stirring can be used to assist dissolution during the dissolution process. When divinyl sulfone is added, its mass concentration in the solution can be controlled to be 3 wt.% to 6 wt.%, such as 3 wt.%, 4 wt.%, 4.5 wt.%, 5 wt.% or 6 wt.%. The concentration of the hydrochloric acid solution can be 0.03 mol / L to 0.06 mol / L, such as 0.03 mol / L, 0.05 mol / L or 0.06 mol / L, and the amount of the hydrochloric acid solution can be controlled to be 4 to 6 times the volume of the cross-linked sodium hyaluronate gel; the stirring reaction is generally carried out at room temperature (about 25°C), and the stirring reaction time can be controlled to be 10h to 15h, such as 10h, 12h, 13h or 15h. The concentration of the phosphate buffer solution can be 0.04 mol / L to 0.06 mol / L, the pH value is 7.0, and the amount of the phosphate buffer solution can be 45 to 55 times the volume of the cross-linked sodium hyaluronate gel. The mixing process of the gel and the collagen aqueous solution can be assisted by mechanical stirring, and the stirring mixing time can be controlled to be 20h to 30h, such as 20h, 22h, 24h, 28h or 30h.

[0048] In some embodiments of the present invention, step S21 includes preparing chitosan gel containing collagen using functional coating layer raw materials including collagen and chitosan as coating slurry.

[0049] Specifically, collagen can be dissolved in water to obtain a collagen aqueous solution; chitosan is added into water and stirred to be evenly dispersed, and glacial acetic acid is added thereto while stirring to prepare a chitosan aqueous solution; the collagen aqueous solution and the chitosan aqueous solution are stirred and blended, and genipin is added thereto while stirring; stirring is then turned off to carry out a cross-linking reaction at room temperature to obtain a coating slurry.

[0050] Among them, the process of dissolving collagen in water is generally carried out at room temperature (about 25°C); the dissolution process can be assisted by stirring, and the stirring time can be controlled at 5min to 8min; after the stirring is completed, the solution can be allowed to stand for defoaming treatment, and the standing time can be controlled at 20min to 40min; the concentration of the prepared collagen aqueous solution can be controlled to be 0.005g / mL to 0.015g / mL, such as 0.005g / mL, 0.01g / mL, 0.012g / mL or 0.015g / mL.

[0051] The process of preparing the chitosan aqueous solution is generally carried out at room temperature (about 25° C.), and the stirring and dispersing time can be controlled to be 5h to 7h, such as 5h, 6h, 6.5h or 7h; the mass fraction of the added glacial acetic acid can be controlled to be 0.2wt.% to 0.4wt.%, such as 0.2wt.%, 0.25wt.%, 0.3wt.% or 0.4wt.%; the concentration of the prepared chitosan aqueous solution can be controlled to be 0.2g / mL to 0.3g / mL, such as 0.2g / mL, 0.25g / mL, 0.28g / mL or 0.3g / mL.

[0052] The collagen aqueous solution and the chitosan aqueous solution can be stirred and blended at a mass ratio of 1:1.2-1.6, and the amount of genipin added during the blending process can be controlled to be 0.01wt.%-0.03wt.%, for example, 0.01wt.%, 0.015wt.%, 0.02wt.% or 0.03wt.%; the stirring and blending time can be controlled to be 30min-60min. The cross-linking reaction time can be controlled to be 20h-30h, for example, 20h, 24h, 26h or 30h.

[0053] In some embodiments of the present invention, step S22 includes, under stirring, adding L-polylactic acid microsphere powder to the coating slurry, stirring and mixing uniformly. Wherein, the amount of L-polylactic acid microsphere powder added can be controlled to be 0.1wt.% to 1wt.%, for example, 0.1wt.%, 0.3wt.%, 0.5wt.%, 0.6wt.%, 0.8wt.% or 1wt.%; the stirring and mixing time can be controlled to be 1h to 6h, for example, 1h, 2h, 4h, 5h or 6h. The stirring state is generally a stirring state at room temperature (about 25°C).

[0054] In some embodiments of the present invention, before step S22, the process further includes: preparing L-polylactic acid microsphere powder, which specifically includes:

[0055] 1) polyvinyl alcohol, a surfactant (such as Tween 80) and deionized water are mixed to obtain an aqueous phase W; a surfactant (such as tributyl citrate) and a biodegradable polyester are mixed in dichloromethane to obtain an oil phase O;

[0056] 2) adding the oil phase O to the water phase W and homogenizing to obtain an O / W emulsion;

[0057] 3) Mechanically stirring the O / W emulsion to form polyester microspheres; then collecting the L-polylactic acid microspheres by centrifugation, washing and freeze-drying to obtain L-polylactic acid microsphere powder.

[0058] The size, shape and surface smoothness of the L-polylactic acid microspheres in the above medical materials have a great influence on tissue reaction and determine the degree of reaction to a certain extent. The above method can be used to prepare L-polylactic acid microspheres with a smooth surface, regular spherical shape and uniform particle size, which can greatly reduce the occurrence of inflammation after implantation; and the high surface smoothness of the microspheres can be conducive to subcutaneous injection, can stimulate the fibroblasts under the wrinkles to wrap one by one, and can also reduce the occurrence of foreign body reaction.

[0059] In some embodiments of the present invention, in step 1), during the preparation of the aqueous phase W, 2 mg to 20 mg of polyvinyl alcohol and 0.5 mg to 5 mg of a surfactant are added per milliliter of deionized water. The amount of polyvinyl alcohol added may be 2 mg, 4 mg, 5 mg, 8 mg, 10 mg, 12 mg, 15 mg, 18 mg, 20 mg, etc.; the amount of the surfactant added may be 0.5 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 3.5 mg, 4 mg, 5 mg, etc.

[0060] In some embodiments of the present invention, the biodegradable polyester may be a copolymer of one or more of polyglycolic acid, polylactic acid, polycaprolactone, and polydioxanone.

[0061] In some embodiments of the present invention, the content of biodegradable polyester in the oil phase O is 2.5wt.% to 20wt.%, for example, 2.5wt.%, 4wt.%, 5wt.%, 8wt.%, 10wt.%, 12wt.%, 15wt.%, 17wt.% or 20wt.%.

[0062] In some embodiments of the present invention, the amount of surfactant added per milliliter of oil phase O is 0.5 mg to 5 mg, for example, 0.5 mg, 0.8 mg, 1 mg, 2 mg, 2.5 mg, 3 mg, 4 mg, 4.5 mg, 5 mg, etc.

[0063] In some embodiments of the present invention, in step 2), the mass ratio of the water phase W to the oil phase O can be controlled to be 3:1 to 1:1, for example, 3:1, 2.5:1, 2:1, 1.8:1, 1.5:1 or 1:1.

[0064] In some embodiments of the present invention, in step 2), the homogenization speed can be controlled at 2000 rpm to 10000 rpm, and the time can be controlled at 5 min to 15 min. Further, the homogenization speed can be controlled at 5000 rpm to 10000 rpm, such as 5000 rpm, 6000 rpm, 6500 rpm, 7000 rpm, 8000 rpm, 8500 rpm, 9000 rpm or 10000 rpm. The homogenization time can be 5 min, 6 min, 8 min or 10 min.

[0065] In some embodiments of the present invention, in step 3), the speed of mechanical stirring is 100 rpm to 500 rpm, for example, 100 rpm, 200 rpm, 250 rpm, 300 rpm, 400 rpm, 450 rpm or 500 rpm; the time of mechanical stirring can be 1 h to 10 h, such as 1 h, 3 h, 5 h, 6 h, 8 h or 10 h.

[0066] In some embodiments of the present invention, in step 3), the centrifugal speed can be controlled at 3500 rpm to 4500 rpm, such as 3500 rpm, 3600 rpm, 3800 rpm, 4000 rpm or 4500 rpm; the centrifugal time can be 20 min to 40 min, such as 20 min, 25 min, 30 min or 40 min. The washing treatment can be specifically washed with deionized water for 3 to 6 times; the freeze-drying time can be controlled at 24 h to 48 h.

[0067] In some embodiments of the present invention, in step S22, before mixing the L-polylactic acid microsphere powder with the coating slurry, the surface of the L-polylactic acid microspheres in the L-polylactic acid microsphere powder is loaded with an adsorption-promoting layer. Specifically, the surface of the L-polylactic acid microspheres in the L-polylactic acid microsphere powder is loaded with an adsorption-promoting layer to obtain a L-polylactic acid microsphere powder with a surface loaded adsorption-promoting layer, and then the L-polylactic acid microsphere powder with a surface loaded adsorption-promoting layer is mixed with the coating slurry.

[0068] In some embodiments of the present invention, a polyethyleneimine layer can be coupled and loaded on the surface of the L-polylactic acid microspheres by the carbodiimide method as an adsorption promoting layer.

[0069] In some embodiments of the present invention, a polydopamine layer can be deposited on the surface of the L-polylactic acid microspheres as an adsorption-promoting layer by the dopamine alkaline self-polymerization method. The specific method may include: mixing dopamine with water to prepare a dopamine aqueous solution with a concentration of 0.5 mg / mL to 5 mg / mL, and using a buffer to adjust the pH range to 7.2 to 9; suspending the L-polylactic acid microsphere powder in the dopamine aqueous solution, reacting at room temperature for 12h to 48h, and then washing and freeze-drying to obtain the L-polylactic acid microsphere powder with a polydopamine layer on the surface. Among them, the buffer can be PBS buffer or MES buffer; washing can be done with deionized water, and specifically can be washed 2 to 5 times, for example, 2 times, 3 times or 5 times.

[0070] In some embodiments of the present invention, a calcium phosphate layer can be deposited on the surface of the poly(L-lactic acid) microspheres as an adsorption-promoting layer by a biomineralization method.

[0071] In some embodiments of the present invention, the preparation method further comprises: S23, packaging and sterilization.

[0072] In some embodiments of the present invention, in step S21, a collagen aqueous solution is prepared as a coating slurry, and the corresponding medical material product type obtained is freeze-dried microspheres; further, in step S23, the packaging method can be pharmaceutical bottle packaging.

[0073] In some embodiments of the present invention, in step S21, a collagen-containing gel is prepared as a coating slurry, and the corresponding medical material product type obtained is a gel; further, in step S23, the packaging method can be injection packaging.

[0074] In some embodiments of the present invention, in step S23, the sterilization method of the sterilization treatment is γ-ray irradiation sterilization or β-ray irradiation sterilization. The dose of γ-ray irradiation can be 8kGy to 16kGy, such as 8kGy, 10kGy, 12kGy, 15kGy or 16kGy; the dose of β-ray irradiation is 6kGy to 10kGy, such as 6kGy, 7kGy, 9kGy or 10kGy. DETAILED DESCRIPTION

[0075] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0076] The experimental materials used in the following embodiments of the present invention include:

[0077] (1) Biodegradable polyesters: polycaprolactone (PCL), molecular weight 80,000; polylactic acid-glycolic acid copolymer (PLGA), molecular weight 500,000; polyglycolic acid (PGA), molecular weight 130,000; polylactic acid-caprolactone copolymer (PLCL), molecular weight 150,000; poly (L-lactic acid) (PLLA), molecular weight 240,000.

[0078] (2) Chemical and biological reagents: All were purchased from MedChemExpress and Sigma-Aldrich reagent websites.

[0079] (3) The proliferation-promoting culture medium kit used in the example, brand: BI, item number: 05-200-1B; the basic culture medium is DMEM, brand: Gibco, item number: 12430054.

[0080] (4) If no specific conditions are specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0081] Example 1

[0082] This embodiment proposes a regenerated human type I collagen, the preparation method of which comprises the following steps:

[0083] S1. Human skin cells or mesenchymal stem cells are isolated and cultured in vitro using tissue engineering methods.

[0084] S2. The cells were inoculated into a custom-designed tissue culture apparatus for culture. The custom-designed tissue culture apparatus was a 20-layer cuboid container treated with adherent TC. The total culture time was controlled to be 22 days.

[0085] Specifically, the initial cell seeding density was 8000 / cm 2 , first statically cultured for 4 days, the culture medium is a proliferation-promoting culture medium kit, and the cell fusion degree is observed under a microscope to reach more than 95%. Then it is replaced with a secretory protein culture medium for dynamic culture. The composition of the secretory protein culture medium is a basal culture medium and factors, wherein the basal culture medium is DMEM / MEM (i.e., a composite of two culture media with a mass ratio of 1:1); the factors include 25ug / mL ascorbic acid phosphate magnesium salt, 36mg / mL of glycine, alanine, proline, lysine, hydroxyproline and methionine with a mass ratio of 1:1:1:1:1:1, 0.025ug / mL betamethasone, 0.025ug / mL insulin transferrin, and 3wt.% fetal bovine serum or serum substitute. During the dynamic culture process, the secretory protein culture medium was replaced every 4 days. At the same time, the circulation of the culture medium was controlled from the 4th day of culture to stimulate the circulating flow of the culture medium. The flow rate of the first liquid change was 7rpm. The pump speed was increased to 20rpm for the second liquid change and to 36rpm for the third liquid change to promote the secretion of collagen and tissue formation.

[0086] S3. After the total culture time reaches 22 days, the cells are subjected to in situ decellularization to collect collagen. The eluent for in situ decellularization consists of 1.8 mM sodium dodecyl sulfate (SDS), 5 mM ethylenediaminetetraacetic acid (EDTA), and 1 M sodium chloride (NaCl); the specific implementation method is to use the eluent for in situ circulation perfusion for 6 hours, and then use PBS buffer solution for circulation perfusion 3 times, each time for 15 minutes, to obtain regenerated human type I collagen.

[0087] In the above preparation method, during the cell culture process, after static proliferation culture, it is replaced with a secretion-promoting protein culture medium containing specific factors. Collagen is mainly composed of the above 6 kinds of amino acids. The stimulation of the three factors of ascorbic acid phosphate magnesium salt, betamethasone, and insulin transferrin on cells can better promote amino acid synthesis and secretion of collagen. Among them, betamethasone can effectively inhibit the proliferation and division of cells, making them tend to secrete proteins. Factor ascorbic acid phosphate magnesium salt and factor insulin transferrin can effectively promote cell secretion of collagen. After replacing with secretion-promoting culture medium, the culture medium circulation system is turned on for dynamic culture. At this time, the cells are basically no longer as rapidly proliferating and dividing as static culture, and basically maintain their existing cell mass. Under the stimulation of secretion-promoting culture medium and circulating dynamic culture medium, the cells begin to secrete collagen in large quantities, and show a superimposed secretion effect, thereby increasing the yield of collagen. In addition, the specific operation of the above preparation method is controllable, and the batch differences are almost eliminated, which is conducive to ensuring the uniformity and stability of mass production; and the product collagen is derived from humans, which can effectively eliminate the risks of immunogenicity and viral residues in animal-derived collagen, and has strong safety and reliability.

[0088] Comparative Example 1

[0089] This comparative example proposes a regenerated human type I collagen, and its preparation method is different from that of Example 1 in that: in this comparative example, the addition of the factors insulin transferrin is omitted in the secretory protein culture medium, and other components, dosages and operations are the same as those in Example 1.

[0090] Comparative Example 2

[0091] This comparative example proposes a regenerated human type I collagen, and its preparation method is different from that of Example 1 in that: in this comparative example, the addition of the factor magnesium ascorbyl phosphate is eliminated in the secretory protein culture medium, and other components, dosages and operations are the same as those in Example 1.

[0092] Comparative Example 3

[0093] This comparative example proposes a regenerated human type I collagen, and its preparation method is different from that of Example 1 in that: in this comparative example, the addition of the factors magnesium ascorbyl phosphate and insulin transferrin is omitted in the secretory protein culture medium, and the other components, dosages and operations are the same as those in Example 1.

[0094] Preparation method effect test

[0095] Detection of collagen content: First detect the content of total protein and impurity protein in the sample, and then calculate the collagen content in the sample by indirect methods.

[0096] Among them, the method for determining the content of impure proteins is as follows: take a sample and add a rated volume of purified water according to different specifications, shake it until it is completely dissolved, let it stand until the bubbles disappear, and take the solution and determine it according to Appendix B "Determination of the total amount of impure proteins" of YY 0954-2015 "Passive Surgical Implant Type I Collagen Implant".

[0097] Method for determining total protein: Take the extracted collagen and add the rated volume of purified water according to different specifications, shake until completely dissolved, let stand until the bubbles disappear, and take the solution to determine the total protein content in the sample according to the "Third Method Biuret Method" in the protein content determination method of the "Pharmacopoeia of the People's Republic of China (Volume 4)" (2020 edition).

[0098] The collagen content in the sample was calculated according to the following formula: collagen content in the sample = total protein content * (1-miscellaneous protein content).

[0099] The above method was used to test the collagen content and yield in the products obtained by the preparation method of regenerated human type I collagen in each embodiment and comparative example. The results are shown in Table 1 below.

[0100] Table 1

[0101]

[0102] From Table 1 above, it can be seen that among the three factors of ascorbyl phosphate magnesium salt, betamethasone, and insulin transferrin, ascorbyl phosphate magnesium salt and insulin transferrin can effectively promote the secretion of collagen by cells, and the lack of any one of them will affect the collagen yield. When both are lacking, the yield drops to 3.5%. Therefore, the combination of the three factors in the secretory protein culture medium is essential.

[0103] Example 2

[0104] This embodiment provides a medical material, which includes L-polylactic acid microspheres and a surface functional coating layer loaded on the L-polylactic acid microspheres, wherein the functional coating layer is a sodium alginate gel layer containing collagen. The collagen in the functional coating layer is the regenerated human type I collagen prepared in Example 1.

[0105] The preparation method of the medical material comprises the following steps:

[0106] S1, preparing L-polylactic acid microsphere powder, specifically comprising:

[0107] a. 0.8 g polyvinyl alcohol, 0.1 g surfactant Tween 80 and 100 mL deionized water were mixed to obtain an aqueous phase W; 32 mg surfactant tributyl citrate and 2.12 g L-polylactic acid were added to 40 mL dichloromethane to obtain an oil phase O.

[0108] b. Add the oil phase O to the water phase W, with the ratio of water phase to oil phase being 2.5:1, and homogenize at 5000 rpm for 5 min to obtain an O / W emulsion;

[0109] c. The O / W emulsion was mechanically stirred at 200 rpm for 8 h to form L-polylactic acid microspheres. Then the stirring was stopped and the L-polylactic acid microspheres were collected by centrifugation at 4000 rpm for 30 min. After washing with deionized water for 5 times, the microspheres were freeze-dried for 48 hours to obtain L-polylactic acid microsphere powder.

[0110] S2. Prepare coating slurry. Specifically, 5wt.% of the regenerated human type I collagen prepared in Example 1 and 1wt.% of sodium alginate are dissolved and dispersed in water, fully swollen, and 4wt.% of mannitol is added to adjust the osmotic pressure to 300mosm / L to obtain sodium alginate gel containing regenerated human type I collagen as coating slurry.

[0111] S3. Under stirring at room temperature, add 0.5 wt.% of the L-polylactic acid microsphere powder obtained in step S1 to the coating slurry obtained in step S2, and stir for 5 hours to fully mix, so that the surface of the L-polylactic acid microsphere is loaded with a sodium alginate gel layer containing regenerated human type I collagen to obtain a medical material. The product type is gel.

[0112] Example 3

[0113] This embodiment provides a medical material, which includes L-polylactic acid microspheres and a surface functional coating layer loaded on the L-polylactic acid microspheres, wherein the functional coating layer is a sodium hyaluronate gel layer containing collagen. The collagen in the functional coating layer is the regenerated human type I collagen prepared in Example 1.

[0114] The preparation method of the medical material comprises the following steps:

[0115] S1. Prepare L-polylactic acid microsphere powder. The specific operation is the same as step S1 in Example 2.

[0116] S2. Prepare coating slurry. Specifically, dissolve sodium hyaluronate (weight average molecular weight 160000) in 2mol / L sodium hydroxide solution, and control the mass concentration of sodium hyaluronate in the solution to be 20wt.%; after stirring until fully dissolved, add divinyl sulfone, and control the mass concentration of divinyl sulfone in the solution to be 5wt.%; then raise the temperature to 60°C and maintain mechanical stirring for 12h to obtain cross-linked sodium hyaluronate gel; then stop heating, place the obtained cross-linked sodium hyaluronate gel in 0.05mol / L hydrochloric acid solution, and add divinyl sulfone to obtain a cross-linked sodium hyaluronate gel; The added volume of the solution is 5 times the volume of the cross-linked hyaluronic acid gel, and the reaction is stirred at room temperature for 12 hours; the hydrochloric acid solution is removed by suction, and 0.05 mol / L phosphate buffer with a pH value of 7.0 is added to dissolve it. The added volume of the buffer is 50 times the volume of the cross-linked sodium hyaluronate gel; a small amount of water is used to dissolve the regenerated human type I collagen powder and then added to the gel prepared in the above steps, and mechanically stirred for 24 hours until the mixture is uniform, to obtain a sodium hyaluronate gel containing regenerated human type I collagen as a coating slurry.

[0117] S3. Under stirring at room temperature, add 0.5 wt.% of the L-polylactic acid microsphere powder obtained in step S1 to the coating slurry obtained in step S2, and stir for 5 hours to fully mix, so that the surface of the L-polylactic acid microsphere is loaded with a sodium hyaluronate gel layer containing regenerated human type I collagen to obtain a medical material. The product type is gel.

[0118] Example 4

[0119] This embodiment provides a medical material, which includes L-polylactic acid microspheres and a surface functional coating layer loaded on the L-polylactic acid microspheres, wherein the functional coating layer is a chitosan gel layer containing collagen. The collagen in the functional coating layer is the regenerated human type I collagen prepared in Example 1.

[0120] The preparation method of the medical material comprises the following steps:

[0121] S1. Prepare L-polylactic acid microsphere powder. The specific operation is the same as step S1 in Example 2.

[0122] S2. Prepare coating slurry. Specifically, at room temperature, take 0.5g of regenerated human type I collagen obtained in Example 1 and add it to 50mL of deionized water, place it on a magnetic stirrer and stir it thoroughly for 8min to dissolve, and let it stand for 30min to eliminate foam to obtain a collagen aqueous solution; at room temperature, add 0.8g of chitosan to 30mL of deionized water, place it on a magnetic stirrer and disperse it evenly, add 0.3wt.% of glacial acetic acid while stirring, adjust the speed and stir it for 6h to obtain a chitosan aqueous solution; blend the collagen aqueous solution and the chitosan aqueous solution in a mass ratio of 1:1.5, place it on a magnetic stirrer and stir it for 50min, and then add 0.02wt.% of genipin and continue stirring for 30min; then turn off the stirring and perform a cross-linking reaction at room temperature for 24h to obtain a chitosan gel containing regenerated human type I collagen as a coating slurry.

[0123] S3. Under stirring at room temperature, add 0.5 wt.% of the L-polylactic acid microsphere powder obtained in step S1 to the coating slurry obtained in step S2, and stir for 5 hours to fully mix, so that the surface of the L-polylactic acid microsphere is loaded with a chitosan gel layer containing regenerated human type I collagen to obtain a medical material. The product type is gel.

[0124] Example 5

[0125] This embodiment provides a medical material, which includes L-polylactic acid microspheres and a surface functional coating layer loaded on the L-polylactic acid microspheres, wherein the functional coating layer is a collagen layer. The collagen in the collagen layer is the regenerated human type I collagen prepared in Example 1.

[0126] The preparation method of the medical material comprises the following steps:

[0127] S1. Prepare L-polylactic acid microsphere powder. The specific operation is the same as step S1 in Example 2.

[0128] S2. Prepare coating slurry. Specifically, at room temperature, take 0.1 g of regenerated human type I collagen prepared in Example 1 and add it to 100 mL of deionized water, place it on a magnetic stirrer and stir it thoroughly for 60 minutes to dissolve, and let it stand for 30 minutes to eliminate foam, to obtain a collagen aqueous solution as coating slurry.

[0129] S3. Under normal temperature stirring, add 0.5 wt.% of the L-polylactic acid microsphere powder obtained in step S1 to the coating slurry obtained in step S2, and stir for 5 hours to fully mix, so that the surface of the L-polylactic acid microspheres is loaded with a regenerated human type I collagen layer to obtain a medical material. The product type is freeze-dried microspheres.

[0130] Example 6

[0131] This embodiment provides a medical material, which differs from Embodiment 2 in that: the medical material of this embodiment further includes an adsorption promoting layer, which is a polydopamine layer, specifically arranged between the left-handed polylactic acid microspheres and the functional coating layer (i.e., the sodium alginate gel layer containing regenerated human type I collagen); other structures are the same as the medical material in Embodiment 2.

[0132] The preparation method of the medical material comprises:

[0133] S1, first prepare L-polylactic acid microsphere powder by the same method as step S1 in Example 2, and then load the adsorption-promoting layer on the surface of the L-polylactic acid microspheres in the L-polylactic acid microsphere powder, specifically comprising: preparing a 2 mg / mL dopamine aqueous solution, adjusting the pH range to 8.5 with a PBS buffer, suspending the L-polylactic acid microsphere powder therein, reacting at room temperature for 24 hours, washing with deionized water 3 times, freeze-drying, forming a polydopamine layer on the surface of the L-polylactic acid microsphere as the adsorption-promoting layer, and obtaining a L-polylactic acid microsphere powder with a polydopamine layer on the surface;

[0134] S2. Prepare coating slurry according to the same operation as step S2 in Example 2;

[0135] S3, using the L-lactic acid microsphere powder loaded with a polydopamine layer on the surface obtained in step S1 to replace the L-lactic acid microsphere powder in Example 2, and other operations are the same as step S3 in Example 2.

[0136] Example 7

[0137] This embodiment provides a medical material, which differs from Embodiment 3 in that: the medical material of this embodiment further includes an adsorption promoting layer, which is a polydopamine layer, specifically arranged between the left-handed polylactic acid microspheres and the functional coating layer (i.e., the sodium hyaluronate gel layer containing regenerated human type I collagen); other structures are the same as the medical material in Embodiment 3.

[0138] The preparation method of the medical material comprises:

[0139] S1. Prepare L-lactic acid microsphere powder with a surface-loaded polydopamine layer by the same operation as step S1 in Example 6.

[0140] S2. Prepare coating slurry according to the same operation as step S2 in Example 3;

[0141] S3, using the L-lactic acid microsphere powder loaded with a polydopamine layer on the surface obtained in step S1 to replace the L-lactic acid microsphere powder in Example 3; other operations are the same as in Example 3.

[0142] Example 8

[0143] This embodiment provides a medical material, which differs from Embodiment 4 in that: the medical material of this embodiment further includes an adsorption promoting layer, which is a polydopamine layer, which is specifically arranged between the left-handed polylactic acid microspheres and the functional coating layer (i.e., the chitosan gel layer containing regenerated human type I collagen); other structures are the same as the medical material in Embodiment 4.

[0144] The preparation method of the medical material comprises:

[0145] S1. Prepare L-lactic acid microsphere powder with a surface-loaded polydopamine layer by the same operation as step S1 in Example 6.

[0146] S2. Prepare coating slurry according to the same operation as step S2 in Example 4;

[0147] S3, using the L-lactic acid microsphere powder loaded with a polydopamine layer on the surface obtained in step S1 to replace the L-lactic acid microsphere powder in Example 4; other operations are the same as in Example 4.

[0148] Example 9

[0149] This embodiment provides a medical material, which differs from Embodiment 5 in that: the medical material of this embodiment also includes an adsorption promoting layer, which is a polydopamine layer, specifically arranged between the left-handed polylactic acid microspheres and the functional coating layer (i.e., the regenerated human type I collagen layer); the other structures are the same as the medical material in Embodiment 5.

[0150] The preparation method of the medical material comprises:

[0151] S1. Prepare L-lactic acid microsphere powder with a surface-loaded polydopamine layer by the same operation as step S1 in Example 6.

[0152] S2. Prepare a coating slurry in the same manner as step S2 in Example 5;

[0153] S3, using the L-lactic acid microsphere powder loaded with a polydopamine layer on the surface obtained in step S1 to replace the L-lactic acid microsphere powder in Example 5; other operations are the same as in Example 5.

[0154] Comparative Example 4

[0155] In this comparative example, the L-polylactic acid microsphere powder obtained in step S1 of Example 2 is directly used as the medical material.

[0156] Comparative Example 5

[0157] In this comparative example, the surface-loaded polylactic acid (L-polylactic acid) microsphere powder obtained in step S1 of Example 6 is directly used as the medical material.

[0158] Comparative Example 6

[0159] This comparative example proposes a medical material. The difference between this comparative example and Example 8 is that this comparative example uses commercially available bovine type I collagen to replace the regenerated human type I collagen used in Example 1 in Example 8. The manufacturer of the bovine type I collagen is Zhejiang Chongshan Biological Products Co., Ltd., and the model is type I active collagen freeze-dried powder; the rest of the medical material and its preparation method are the same as those in Example 8.

[0160] Comparative Example 7

[0161] This comparative example proposes a medical material. The difference between this comparative example and Example 8 is that this comparative example uses commercially available recombinant type III collagen to replace the regenerated human type I collagen used in Example 1 in Example 8. The recombinant type III collagen is specifically purchased from Shanxi Jinbo Biopharmaceutical Co., Ltd., and the model is Recombinant type III humanized collagen freeze-dried fiber (mechanical); otherwise the same as the medical material and preparation method thereof in Example 8.

[0162] When the medical materials of the above embodiments and comparative examples are used, the above medical materials for facial subcutaneous injection can be used after being compounded with injection water.

[0163] The above examples and comparative examples of medical materials were used to test the facial filling and repair performance of volunteers in some medical beauty clinics. The results are shown in Table 2 below.

[0164] Table 2

[0165]

[0166] From the test results of the medical materials of Examples 2 to 9 and Comparative Examples 4 to 7, it can be seen that the filling effect of the left-handed polylactic acid microspheres in Comparative Example 4, which are not loaded with an adsorption-promoting layer on the surface, is poor, and after filling, they will collapse rigidly and have a short duration. The filling effect of the left-handed polylactic acid microspheres in Comparative Example 5, which are loaded with a polydopamine layer as an adsorption-promoting layer on the surface, is poor, and the duration is slightly longer than that of Comparative Example 4. Compared with Comparative Example 4, the medical materials of Examples 2 to 5 are combined with regenerated human type I collagen by using left-handed polylactic acid microspheres that are not loaded with an adsorption-promoting layer on the surface, which can improve the filling effect and duration of the material. However, since the surface of the left-handed polylactic acid microspheres is not loaded with an adsorption-promoting coating, the compositeness of collagen and the left-handed polylactic acid microspheres is relatively poor, and they are prone to separation after implantation in the human body, resulting in slight stiffness, affecting the long-term implantation effect. In the medical materials of Examples 6 to 9, there is a polydopamine layer as an adsorption-promoting layer between the L-polylactic acid microspheres and the functional coating layer containing regenerated human type I collagen, which can effectively composite the functional coating layer and the L-polylactic acid microspheres, improve the composite stability of the material, ensure that the material has no side effects after being implanted in the human body, and can be maintained for a long time. The medical material of Comparative Example 6 is a bovine collagen product, which will be rejected and cause stiffness after being implanted in the human body; and the medical material of Comparative Example 7 is a recombinant collagen product, which will degrade quickly after being implanted in the human body, and the long-term implantation effect is poor.

[0167] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing regenerated human type I collagen, characterized in that: The following steps are involved: S11. Isolating and proliferating human skin cells or mesenchymal stem cells in vitro; S12, inoculating the cells into proliferation medium for static culture; Then, dynamic culture is carried out in a secretagogue protein culture medium; the secretagogue protein culture medium includes a basal culture medium and factors; the factors in the secretagogue protein culture medium include 20ug / mL to 60ug / mL of ascorbyl phosphate magnesium salt, 0.02ug / mL to 0.05ug / mL of glucocorticoid, and 0.02ug / mL to 0.06ug / mL of insulin transferrin.

2. The method for preparing regenerated human type I collagen according to claim 1, characterized in that: The factors in the secretagogue protein culture medium further include: 30 mg / mL to 70 mg / mL of glycine, alanine, proline, lysine, hydroxyproline and methionine in a mass ratio of 1:1:1:1:1:1, and / or 2 wt.% to 10 wt.% of fetal bovine serum or serum substitute.

3. The method for preparing regenerated human type I collagen according to claim 1, characterized in that: In step S12, the cells are inoculated into the proliferation medium at an initial density of 6000 to 20000 cells / cm 2 ; and / or, the static culture time is 2 to 4 days.

4. The method for preparing regenerated human type I collagen according to claim 1, characterized in that: In step S12, the basal culture medium is selected from DMEM culture medium and / or MEM culture medium; and / or the glucocorticoid is selected from at least one of betamethasone, methylprednisolone, dexamethasone and beclomethasone.

5. The method for preparing regenerated human type I collagen according to claim 1, characterized in that: In step S12, the dynamic culture is a cyclic dynamic culture, including: replacing the culture medium every 2 to 7 days, and controlling the circulation flow of the culture medium from the 2nd to 7th day of the dynamic culture; And / or, the total culture duration of the static culture and the dynamic culture is 13 to 28 days.

6. The method for preparing regenerated human type I collagen according to any one of claims 1 to 5, characterized in that: Also includes: S13. Collect collagen.

7. The method for preparing regenerated human type I collagen according to claim 6, characterized in that: In step S13, collagen is collected by in situ decellularization.

8. A regenerated human type I collagen, characterized in that: The regenerated human type I collagen is prepared by the method for preparing the regenerated human type I collagen according to any one of claims 1 to 7.

9. A medical material, characterized in that: include: L-polylactic acid microspheres and a functional coating layer loaded on the surface of the L-polylactic acid microspheres, wherein the functional coating layer comprises collagen, and the collagen is the regenerated human type I collagen according to claim 8.

10. The medical material according to claim 9, characterized in that: It also includes an adsorption-promoting layer; the adsorption-promoting layer is arranged between the left-handed polylactic acid microspheres and the functional coating layer; And / or, the functional coating layer is a collagen layer or a gel layer containing collagen.

11. The medical material according to claim 10, characterized in that: The adsorption promoting layer is any one of a polyethyleneimine layer, a polydopamine layer, and a calcium phosphate layer; And / or, the gel layer is any one of a sodium alginate gel layer, a sodium hyaluronate gel layer, and a chitosan gel layer.

12. The method for preparing the medical material according to any one of claims 9 to 11, characterized in that: The following steps are involved: S21, preparing a coating slurry using a functional coating layer raw material including collagen; S22, mixing the L-polylactic acid microsphere powder with the coating slurry.

13. The method for preparing the medical material according to claim 12, characterized in that: In step S22, before mixing the L-polylactic acid microsphere powder with the coating slurry, an adsorption promoting layer is first loaded on the surface of the L-polylactic acid microspheres in the L-polylactic acid microsphere powder.

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