Preparation method of compound collagen filler for injectionTechnical Field
The invention belongs to the technical field of medical and medical injection, and particularly relates to a preparation method of a compound collagen filler for injection.
Background
In the aspect of raw material selection of injectable filling, sodium hyaluronate and collagen are mainly used at present, but the materials are easy to be absorbed by body tissues, have short degradation period, have a filling effect of temporarily occupying skin volume and causing limited local host tissue reaction (limited regenerated fibrosis), are difficult to maintain the effects of long-term filling and collagen regeneration induction, and have limited application for providing toxic cross-linking agent components introduced in long-term degradation time.
Because of these problems, several products for tissue repair treatment using biodegradable polymers have been recently developed and used as filler formulations of existing biocompatible polymers, or formulations dispersed into fine particles by a medium having viscosity (CMC, etc.) after treating water-insoluble polymers, such as polycaprolactone filler, etc., which promote regeneration of collagen by stimulation of degradation products to play a repairing role, and since degradation takes a certain time, immediate effect is insignificant and nutritional effect of CMC is not strong. The collagen has the advantages of single use, poor physical and mechanical properties, single performance, limited application due to unavoidable weaknesses of strong hydrophilicity, easy degradation by collagenase in vivo and the like, the collagen crosslinked by chemical agents can increase the degradation time of the gel but has certain toxicity, polyester filler can not achieve the effect of stimulating the collagen in real time, and the adhesive CMC gel has the filling effect, but is not a human body component and has no nutrition regeneration effect, and the hyaluronic acid gel also has residual toxic effect after crosslinking.
CN118649289a discloses a collagen implant, which performs physical crosslinking, chemical crosslinking and irradiation crosslinking on collagen in sequence to obtain collagen fibers, and performs dispersion emulsification in solution mixing to obtain the collagen implant. In order to increase the crosslinking degree, the patent uses three crosslinking modes, and the triple crosslinking is that the product has high crosslinking degree and low enzymolysis rate, so that the filling effect is more durable. However, chemical crosslinking inevitably introduces chemical crosslinking agents, such as dialdehydes, carbodiimides, etc., and the residue of the crosslinking agent brings more risk of sensitization and even risk of teratogenesis to clinical use.
CN117618671a discloses a collagen filler having an average pore size D50 of 50 μm to 500 μm, a porosity of 80% -99.5%, a degree of crosslinking of 10% to 70%, and an in vitro enzymolysis loss rate of less than 40%. The patent controls the relative distance of collagen fiber bundles in the crosslinking process by controlling the pore size by taking microspheres as templates, thereby controlling the pore size of the collagen fiber bundles after forming a crosslinked framework, and on the other hand, the dynamic single-template (or multi-template) crosslinking process is realized by controlling the mechanical stirring and microsphere adding modes, so that each collagen fiber bundle can obtain the same crosslinking opportunity, and the more thorough and uniform crosslinking effect of the collagen implant is realized.
CN117582549a discloses an injection type recombinant collagen soft tissue filling gel, which comprises, by weight, 5% -10% of a type III recombinant collagen solution, 2% -3% of an injection sodium hyaluronate solution, 1% -3% of medicinal glycerol, 1% -5% of medical hydroxyapatite, and the balance of water. The injection type recombinant collagen soft tissue gel prepared by mixing the III type recombinant collagen solution, the sodium hyaluronate solution for injection, the medicinal glycerol and the medical hydroxyapatite utilizes the screened core functional region fragment with high activity and high water solubility, which is 100 percent the same as the corresponding fragment of human collagen, eliminates immunogenicity, and has the characteristics of high biocompatibility, high bioactivity, high water solubility and no virus reaction.
However, the above prior art has a product in which collagen and microspheres for injection and filling are compounded, but the long-term filling effect cannot be achieved, and the risk of collagen allergy is high.
Disclosure of Invention
In order to solve the defects of short acting time, poor filling effect and short duration of a filler containing collagen, the invention provides a collagen filler for injection, and the filling strength and the duration are increased by crosslinking the collagen and adding microspheres for injection and filling, in particular, the invention provides the following technical scheme for realizing the purposes:
The preparation method of the collagen filling agent for injection comprises the following steps:
(S1) salting out and precipitating the extracted collagen solution, dialyzing the precipitated precipitate, and freeze-drying to obtain collagen;
(S2) dissolving collagen with hydrochloric acid, and homogenizing with PBS buffer solution to prepare non-crosslinked collagen gel;
(S3) dissolving collagen with hydrochloric acid, homogenizing with PBS buffer solution, irradiating the obtained collagen gel by adopting a gradient irradiation crosslinking mode, granulating by using a screen after irradiation to obtain crosslinked collagen gel, wherein the irradiation intensity is gradually increased from 10-15kgy to 25-30kgy, and then maintaining the irradiation intensity;
(S4) mixing the uncrosslinked collagen gel obtained in the step (S2), the crosslinked collagen gel obtained in the step (S3) with microspheres for injection and filling, and degassing and filling to obtain the collagen filler for injection.
Further, in step (S1), collagen sources include, but are not limited to, animal root achilles tendons, such as bovine achilles tendons, porcine achilles tendons, animal skins, such as cow skin, pig skin, cow pericardium, arachnoid membrane.
Further, when the non-crosslinked collagen is prepared in the step (S2), the collagen is derived from animal skins such as cow leather and pig skin, and when the non-crosslinked collagen is prepared in the step (S3), the collagen is derived from animal achilles tendons such as cow achilles tendon and pig achilles tendon, preferably cow achilles tendon. The bovine achilles tendon collagen is more compact, the strength of the formed crosslinked collagen is higher, the collagen content is higher, and the collagen is more suitable for being used as a raw material of the crosslinked collagen. The inventor finds that the non-crosslinked collagen is prepared from collagen derived from animal skin, and the crosslinked collagen is prepared from collagen derived from animal achilles tendon, so that the filling effect of the obtained product collagen filler is optimal.
Further, the concentration of collagen in the non-crosslinked collagen gel is 20-35mg/mL, and the concentration of collagen in the crosslinked collagen gel is 25-32mg/mL. The concentration of the collagen in the crosslinked collagen gel is more strictly limited, the concentration is higher, the yield of the product after water loss can be improved, and the yield can also be improved, but the concentration cannot be too high, otherwise, the strength is too high, the granulation is not easy, the pushing force of the formed filling agent is larger, and the use is inconvenient.
Further, the process for extracting collagen is well known in the art, for example, for collagen derived from animal achilles tendon, after fascia is removed from animal achilles tendon, the animal achilles tendon is washed, frozen, sliced, put into pepsin solution for enzymolysis, centrifugated by enzymolysis solution, salted out supernatant to obtain collagen derived from animal achilles tendon, for collagen derived from animal skin, the hair-removed animal skin is removed, fat layer and epidermis layer are removed, dermis layer is left, water washing, freezing preservation is carried out, soaking in 4-8 DEG CTris-NaCl buffer solution, water washing, enzymolysis is carried out in pepsin solution, residues are removed by centrifugation, and salting out is carried out to obtain collagen derived from animal skin.
Further, when collagen from animal achilles tendon is extracted, freezing temperature is-15 ℃ to 0 ℃, slice thickness is 0.5-2mm, pepsin solution pH is 2-4, the dosage ratio of animal achilles tendon slice to pepsin solution is 1kg:100-200L, pepsin solution concentration is 1g/10L to 1g/30L, enzymolysis time is 3-7 days, salting out and precipitation are sequentially carried out by using saturated NaCl solution, pH=2-3, pH=3-4 and purified water for dialysis for 7-12 days, then collagen from animal achilles tendon is obtained by freeze-drying, when collagen from animal skin is extracted, tris-NaCl buffer solution pH is 7.5-8.5, animal skin concentration in pepsin solution is 1-5g/L, enzymolysis time is 2-5 days, flocculent precipitate obtained by salting out is redissolved in acetic acid solution (acetic acid concentration is 0.1-0.5 mol/L), salting out and redissolved steps are repeated for 2-5 times, and finally salted out and finally, collagen from animal skin is obtained by dialysis in 0.1-0.5M for 2-2 days, namely, collagen from animal skin is obtained by freeze-drying.
Further, the pH of the hydrochloric acid in the step (S2) and the step (S3) is 3-4, and the pH of the PBS buffer solution is=7.2-7.6, such as pH=7.3, pH=7.4 and pH=7.5.
Further, in the step (S2), the ratio of the collagen, the hydrochloric acid and the PBS buffer is 1g:20-30mL:2-3mL.
Further, in the step (S3), the gradient irradiation time is 15-30min, and then the irradiation intensity is maintained for irradiation for 3-5min. The intensity of the gradient irradiation is gradually increased in a nearly linear manner, for example, the irradiation intensity is gradually increased from 10kgy to 30 kgy within 20min, the irradiation intensity increasing rate is about 1kgy/min, and for example, the irradiation intensity is gradually increased from 15kgy to 25ky within 20min, and the irradiation intensity increasing rate is about 0.5kgy/min. Granulating with a screen aperture of 90-150 μm.
Further, in step (S4), injection-filling microspheres are well known in the art, including but not limited to PCL microspheres, PLLA microspheres, PLGA microspheres, and injection-filling microspheres having a particle size of 30-50 μm. Microspheres for injection filling are commercially available or self-made, and the preparation method thereof is well known in the art. For example, for PCL microspheres, a PCL polymer is dissolved by methylene dichloride and/or chloroform to prepare 10-40wt% polymer oil phase solution, PVA is dissolved at 70-90 ℃ to prepare 0.2-2wt% PVA aqueous solution, the aqueous phase and water phase volume ratio is 1:2-10, methylene dichloride/chloroform solvent is volatilized by stirring, and the PCL microspheres with the particle size of 30-50 mu m are prepared by sieving.
Further, in the step (S4), the mass ratio of the uncrosslinked collagen gel, the crosslinked collagen gel, and the microspheres for injection and filling is 2-4:3-5:1-2.
According to the invention, non-crosslinked collagen, crosslinked collagen and degradable polymer microspheres for injection are compounded and mixed according to a certain proportion, so that the instant filling effect can be achieved, the polymer microspheres play a long-term role, and amino acid components of collagen degradation in the early and middle stages can become nutritional substances for stimulating the polyester microspheres to generate collagen regeneration. However, the biggest problem of collagen used for medical injection filling is short duration and anaphylaxis at present, the invention obtains the cross-linked collagen with different cross-linking degrees and the non-cross-linked collagen by compounding with different irradiation intensities, thereby achieving the purposes of long filling duration and good filling effect. The cross-linked collagen of the invention is free from adding any chemical cross-linking agent and toxic action of the chemical agent by irradiation cross-linking technology. The gradient irradiation with gradually increased irradiation intensity can have higher yield and lower water loss under the same concentration. The non-crosslinked collagen gel can provide nutrition restoration microenvironment and filling effect in the early stage, the crosslinked extracellular matrix can provide restoration microenvironment in the middle filling stage, and the degradation of the polyester microspheres in the later stage promotes collagen regeneration, so that the skin has good microenvironment in the whole restoration period, and the skin elasticity is increased.
Drawings
FIG. 1 is a photograph of the crosslinked collagen gel obtained in step S3 of example 1.
FIG. 2 is a photograph of the crosslinked collagen gel obtained in comparative example 1.
FIG. 3 is a photograph of the crosslinked collagen gel obtained in comparative example 2.
FIG. 4 is a photograph of a section of a rabbit subjected to subcutaneous implantation of the filler of example 1 for 12 months.
FIG. 5 is a photograph of a section dyed after 12 months of a subcutaneous implantation experiment of a rabbit with the filler of comparative example 1.
FIG. 6 is a photograph of a section dyed after 12 months of a subcutaneous implantation experiment of rabbits with the filler of comparative example 3.
FIG. 7 is a photograph of a section dyed after 12 months of a subcutaneous implantation experiment of rabbits with the filler of comparative example 3.
Detailed Description
The technical scheme of the invention is further explained by the following specific examples.
Example 1
(S1) extraction of collagen:
(1.1) cutting off the upper bifurcation part of the beef achilles tendon in a Y shape, reserving a trunk part, removing fascia of the beef achilles tendon, washing with purified water for at least 3 times, draining off water, freezing to a refrigerator at (-15-0 ℃) temperature, taking out a cross section slice (thickness of 0.5-2 mm) from the refrigerator, adding the slice into pepsin solution with pH of 2-4 (beef achilles tendon slice: enzymolysis solution of 1 kg/100L-1 kg/200 kg), enzymolysis solution concentration (1 g/10L-1 g/30L), carrying out enzymolysis for 3-7 days, centrifuging to collect supernatant, salting out the supernatant with saturated sodium chloride, dialyzing the separated white floccules in pH=3, pH=4 and purified water for 7 days, and freeze-drying the dialyzate to obtain the collagen of the beef achilles tendon.
(1.2) Selecting fresh pigskin, removing hair and dirt, removing subcutaneous fat layer and epidermis layer by a cutter, leaving dermis layer near the epidermis layer, cutting into size of 2cm multiplied by 2cm, rinsing 3 times by deionized water, preserving the treated pigskin at-20 ℃, immersing the pigskin in Tris-NaCl (Tris: 0.05mol/L; naCl:1mol/L; pH 7.5) buffer solution (feed solution: 1:20) for 12h, rinsing 3 times by deionized water, taking defatted pigskin, hydrolyzing 48h in pepsin solution with pH value of 2-3 at room temperature, centrifuging to remove unreacted residues, adding saturated NaCl into the reacted solution, salting out at 4 ℃ until flocculent precipitate is present in slurry, centrifuging (4 ℃ at 1000 rpm,15 min), collecting lower layer precipitate, redissolving in acetic acid solution with pH value of 2.2 (0.5 mol/L), centrifuging to remove non-protein, dialyzing at pH value of 2-3M, dialyzing at room temperature for 0.0 rpm, dialyzing for 48h, and finally loading the obtained collagen granules into a vacuum dialysis bag, dialyzing for 0.0M for 4h, and freeze-drying the collagen bag.
(S2) preparation of non-crosslinked collagen gel, namely, after 700mg of freeze-dried pigskin collagen is dissolved by 18ml of hydrochloric acid with pH=3, the solution is uniformly mixed with 2ml of PBS to prepare the non-crosslinked collagen gel, wherein the concentration of the collagen is 35mg/ml.
(S3) preparing cross-linked collagen, namely dissolving 1400mg of freeze-dried bovine Achilles tendon collagen by adopting 45ml of hydrochloric acid with pH=3, uniformly mixing the dissolved collagen with 5ml of PBS, preparing gel with the collagen concentration of 28mg/ml, carrying out irradiation cross-linking by adopting cobalt 60, increasing the irradiation intensity from 10kgy to 30kgy at the rate of 1kgy/min within 20min, then continuously carrying out irradiation for 5min, and granulating by adopting a screen mesh of 150 meshes.
(S4) preparation of filler, namely weighing 100g of polycaprolactone, dissolving the polycaprolactone with 250ml of dichloromethane to prepare a polymer oil phase solution, dissolving 3.2g of PVA raw material with 70-90 ℃ of water for injection, cooling to prepare 0.4% (W/V) PVA aqueous solution, mixing the oil phase solution and the PVA aqueous solution in an online shearing machine through a pipeline with a flow pump, shearing and emulsifying at 3200rpm, stirring and volatilizing the dichloromethane, adopting a 30-mu m screen and a 50 mu m screen, and finally freeze-drying to prepare the 30-50 mu m PCL microspheres. Mixing the non-crosslinked collagen gel, the crosslinked collagen gel and the PCL microspheres according to the mass ratio of 3:5:2, and degassing and filling the mixture in a pre-filling and sealing syringe.
Example 2
The other conditions were the same as in example 1, except that in step (S2), the collagen used in step (S3) was pigskin collagen.
Example 3
The other conditions were the same as in example 1, except that in step (S2), the collagen used in step (S3) was bovine Achilles tendon collagen.
Example 4
The other conditions were the same as in example 1, except that the collagen concentration of the non-crosslinked collagen gel in step (S2) was 20mg/mL, and the collagen concentration of the crosslinked collagen gel in step (S2) was 32mg/mL.
Example 5
Other conditions are the same as in example 1, except that in the step (S4), the mass ratio of the non-crosslinked collagen gel, the crosslinked collagen gel, and the PCL microspheres is 4:4:2.
Example 6
Other conditions are the same as in example 1, except that in the step (S4), the mass ratio of the non-crosslinked collagen gel, the crosslinked collagen gel, and the PCL microspheres is 2:3:1.
Example 7
The other conditions were the same as in example 1 except that in step (S3), the collagen concentration of the crosslinked collagen gel was 25mg/mL.
Example 8
Other conditions are the same as in example 1 except that in step (S3), the irradiation intensity is raised from 15kgy to 25kgy at a rate of 0.5kgy/min within 20min, and then irradiation is continued for 5min.
Comparative example 1
The other conditions were the same as in example 1 except that in step (S3), the collagen concentration of the crosslinked collagen gel was 20mg/mL.
Comparative example 2
The other conditions were the same as in example 1 except that in step (S3), irradiation was changed to a fixed irradiation intensity of 20kgy irradiation for 20min.
Comparative example 3
The other conditions are the same as in example 1, except that in the step (S4), the filler is a crosslinked collagen gel and the PCL microspheres are mixed in a mass ratio of 8:2, i.e., no uncrosslinked collagen gel is added.
Comparative example 4
The other conditions are the same as in example 1, except that in the step (S4), the filler is non-crosslinked collagen gel and the PCL microspheres are mixed in a mass ratio of 8:2, i.e., the crosslinked collagen gel is not added.
Application example 1
The bovine achilles tendon collagen and pigskin collagen prepared in step S1 of example 1 were subjected to terminal peptide detection (ELISA), DNA residue (fluorescence method), and α -antigen detection, and the results are shown in table 1 below.
Table 1 residual detection
Analysis for allergen severity, DNA residue > alpha-antigen > terminal peptide, and the animal-derived collagen obtained by the invention has lower immunological reaction.
Application example 2
In the above examples and comparative examples, the mass of the collagen gel before and after crosslinking in step S3 was tested, and the yield after water loss was calculated, and the yield after water loss=the mass of the gel after crosslinking/the mass of the gel before crosslinking. The results are shown in Table 2 below. FIG. 1 is a photograph of the crosslinked collagen gel obtained in step S3 of example 1, FIG. 2 is a photograph of the crosslinked collagen gel obtained in comparative example 1, and FIG. 3 is a photograph of the crosslinked collagen gel obtained in comparative example 2.
Table 2 cross-linked collagen gel yield test
As can be seen, comparative example 1 has a weak gel strength after crosslinking at a concentration of 20mg/ml, is easily broken in the middle, has a low yield (more water loss) after crosslinking, and has a weak elastic modulus (filling property) after being formulated into an injection. The comparative example 1 uses a fixed irradiation intensity, and the yield and the elastic modulus are also somewhat reduced. Whereas example 1 crosslinked collagen was less brittle under compression and more elastic.
Application example 3
The fillers obtained in examples and comparative examples were measured for viscosity and viscoelastic modulus by rheometer, and the degradation and filling effects were observed by subcutaneous implantation experiments using rabbits, and the results are shown in table 3 below. Degradation properties remain with the microspheres and sufficient filling properties are maintained. The injection filler has a viscosity of 0.25Hz within 200-350 Mpa.s, and has good injectability and filling effect. Too low a viscosity indicates a general filling effect, too high a viscosity, difficult injection and easy plugging of the needle.
TABLE 3 filler Effect test
FIG. 4 is a photograph of a section of the filler of example 1 after 12 months of a rabbit subcutaneous implantation test, FIG. 5 is a photograph of a section of the filler of comparative example 1 after 12 months of a rabbit subcutaneous implantation test, FIG. 6 is a photograph of a section of the filler of comparative example 3 after 12 months of a rabbit subcutaneous implantation test, and FIG. 7 is a photograph of a section of the filler of comparative example 3 after 12 months of a rabbit subcutaneous implantation test.