CN115252898B

CN115252898B - Long-acting microparticle type I and type V collagen composite implant

Info

Publication number: CN115252898B
Application number: CN202211001402.4A
Authority: CN
Inventors: 韩淑萍; 何伟; 李贤明; 王雪; 陈文洁
Original assignee: Jiangsu Xihong Biomedical Co ltd
Current assignee: Jiangsu Xihong Biomedical Co ltd
Priority date: 2022-08-19
Filing date: 2022-08-19
Publication date: 2023-08-18
Anticipated expiration: 2042-08-19
Also published as: CN115252898A

Abstract

The invention discloses a microparticle type I and type V collagen composite implant with improved performance and a preparation method thereof. The method comprises the following steps: (1) Preparing a solution, namely an oil phase, of a surfactant and a volatile organic solvent which is not completely dissolved in water, wherein the temperature of the solution is not higher than 4 ℃; (2) Preparing an aqueous solution, namely an aqueous phase, comprising type I and type V collagens and laccase and a protein activity protective agent, at a temperature of not more than 4 ℃; (3) Mixing said aqueous solution with a solution of said organic solvent within 30 minutes after completion of said preparation of said aqueous solution and allowing them to form a water-in-oil emulsion, maintaining the temperature between 4 ℃ and-4 ℃ for more than 24 hours, preferably for less than 48 hours, to substantially crosslink said collagen; (4) Filtering out particles with the particle size of more than 25 mu m and less than 0.5 mu m in the emulsion after the collagen is basically crosslinked, and retaining the particles with the particle size of between 0.5 mu m and 25 mu m; (5) The solvent is removed from the retained particles by cryogenic freezing. The implant has the advantages of long-acting, small fluctuation of the effective period, good needle penetrating property and the like.

Description

Long-acting microparticle type I and type V collagen composite implant

Technical Field

The present invention relates to an injection implant. In particular to a long-acting microparticle type I and type V collagen composite implant with excellent performance.

Technical Field

Type I collagen is the most abundant collagen in humans. It forms large eosinophilic fibers, called collagen fibers. It is present in scar tissue and tissue healing at the end product is through repair, as well as tendons, ligaments, organic parts of the bones of the myofibrils of the intima, in the dermis, dentin and organ capsules.

Extracellular collagen is generally referred to as type v collagen, which is present in large amounts in connective tissue. Type v collagen is distributed around cells and around type i collagen, possibly acting as a bridge between the basement membrane and connective tissue. Some type v collagen is also formed after the skin is wounded or burned and can be used to promote tissue repair.

The combination of the type I collagen and the type V collagen can supplement the missing collagen in situ, and the type I supports the concave part and the type V promotes tissue repair; the filled collagen can be used as attachment and support for cell growth, and can induce the same collagen tissue as host, and stimulate collagen regeneration and repair.

However, the type I and type V collagens have significant disadvantages such as short in vivo residence time.

Although, some long-acting collagen technologies exist, such as:

CN 101648989 (long-acting collagen and method for producing the same): the invention discloses a long-acting collagen and a preparation method thereof, which are characterized in that a pigskin is scraped, grease is removed, swelling, digestion, centrifugal separation, salting out, lower layer sediment collection and freeze drying are carried out to obtain a collagen, then the collagen is mixed with gamma-polyglutamic acid (gamma-PGA), a glutaraldehyde solution is added and uniformly stirred, the first crosslinking is carried out, the glutaraldehyde solution is repeatedly added and uniformly stirred, and the second crosslinking is carried out, thus obtaining the long-acting collagen, which can solve the defects that the residence time of the conventional collagen is too short, frequent supplement and beating are needed, high concentration glutaraldehyde can be remained, the biotoxicity can harm the health of human body, and the like;

CN102924731a (a triple cross-linked collagen and method of manufacture and use): a method for producing triple-crosslinked collagen, comprising: providing a soluble collagen sample; mixing the collagen sample with a first cross-linking agent to form a re-cross-linked collagen; mixing the heavy cross-linked collagen with a second cross-linking agent to form double cross-linked collagen; and mixing the double-crosslinked collagen with a third crosslinking agent to form triple-crosslinked collagen. Wherein the first crosslinking agent, the second crosslinking agent, and the third crosslinking agent are each selected from the group consisting of: the cross-linking agent comprises aldehyde cross-linking agent, imine cross-linking agent and epoxide cross-linking agent, wherein the first cross-linking agent is different from the second cross-linking agent, and the third cross-linking agent is different from the first cross-linking agent and the second cross-linking agent;

however, the above-mentioned techniques use chemical modification methods, which have remarkable effects, but involve the risk of formation of harmful substances during the modification process and the disadvantages of low reaction conditions, production of byproducts, poor specificity, low catalytic efficiency and yield, and loss of collagen during the process.

More importantly, the collagen is cross-linked in a homogeneous phase or cross-linked into a large particle, not a very small particle, and not a substantially uniform particle, but a smaller particle when used, and thus, the apparent therapeutic effect is inconsistent, such as a large fluctuation in the lifespan.

In addition, the preparation is basically gel, and the high viscosity of the aqueous dispersion system causes a plurality of inconveniences in production and use, such as inaccurate dosage and split charging and poor needle penetrating property during injection.

Therefore, there is a need for a composite implant of type I and type v collagen, which has the advantages of long-acting, small fluctuation of the duration, good needle penetration, and the like.

Disclosure of Invention

The invention aims to provide a microparticle type I and type V collagen composite implant with improved performance, long-acting effect, small fluctuation of the effective period and good needle penetration, and a preparation method thereof.

The inventor finds that the combination of the common type I and type V collagen is modified into the microparticle crosslinked collagen with basically uniform size by laccase, so that the action time of the microparticle crosslinked collagen in the body can be prolonged, the fluctuation of the effective period is small, the viscosity of an aqueous dispersion system of the microparticle crosslinked collagen is reduced, various problems caused in the production and use of the microparticle crosslinked collagen are solved, such as accurate split charging of the dosage, and the needle penetrating property is good during injection; in addition, the method can reduce the mixing of harmful substances, make the reaction condition mild, reduce the loss of collagen in the process, improve the specificity, avoid the generation of byproducts, improve the catalytic efficiency, improve the yield and the like.

Laccase (laccase, EC 1.10.3.2) is a copper-containing polyphenol oxidase that catalyzes the oxidation of phenols and aromatic compounds to the corresponding benzoquinone, with the concomitant reduction of molecular oxygen to water by electron transfer. Laccase has a broad substrate specificity, and its substrates include monophenols, diphenols, polyphenols, benzenethiols, aminophenols, anilines, polyamines, lignin aromatic diamines, etc. [18]. Tyrosinase-catalyzed crosslinking is based on quinone formation, whereas laccase-catalyzed crosslinking reactions are based on free radicals and their further reactions.

Based on this, the present invention has been completed.

The invention relates to a (performance improved) long-acting and fine particle type I and type V collagen composite implant with small fluctuation of effective period and good needle penetration, which comprises the following components: the size of the cross-linked type I and type V collagen composite particles is between 0.5 and 25 μm.

The invention relates to a preparation method of a microparticle type I and type V collagen composite implant with improved performance, long-acting, small fluctuation of effective period and good needle penetration, which comprises the following steps:

(1) Preparing a solution, namely an oil phase, of a surfactant and a volatile organic solvent which is not completely dissolved in water, wherein the temperature of the solution is not higher than 4 ℃;

(2) Preparing an aqueous solution, namely an aqueous phase, comprising type I and type V collagens and laccase and a protein activity protective agent, at a temperature of not more than 4 ℃;

(3) Mixing said aqueous solution with a solution of said organic solvent within 30 minutes after completion of said preparation of said aqueous solution and allowing them to form a water-in-oil emulsion, maintaining the temperature between 4 ℃ and-4 ℃ for more than 24 hours, preferably for less than 48 hours, to substantially crosslink said collagen;

(4) Filtering out particles with the particle size of more than 25 mu m and less than 0.5 mu m in the emulsion after the collagen is basically crosslinked, and retaining the particles with the particle size of between 0.5 mu m and 25 mu m;

(5) The solvent is removed from the retained particles by cryogenic freezing.

The meaning of "composite" in the above text corresponds to "combination", and both are equivalent.

Detailed Description

Such protein activity protectants include, but are not limited to, sorbitol, mannitol, erythritol, maltitol, lactitol, xylitol, and combinations thereof. The protein active agent is used for protecting the activities of type I and type V collagen and laccase.

Preferably, the total mass concentration of the above aqueous solution, i.e. the type I and type v collagen in the aqueous phase, is 0.1% to 10%, more preferably 0.5% to 5%; preferably, the ratio of the amount of laccase to the total amount of type I and type V collagen is 1-10U/g, preferably 4-8U/g, more preferably 5-6U/g.

The dosage ratio of the type I collagen to the type V collagen is 8:1 to 1:8, preferably 5:1.

the above organic solvents include, but are not limited to, esters of C1-C6 acids with C1-C6 alcohols, such as ethyl acetate, methyl acetate, ethyl propionate, butyl acetate, pentyl valerate, isopentyl isovalerate, and the like; ethers of C1-C6 alcohols with C1-C6 alcohols, such as diethyl ether, methylethyl ether; C3-C6 ketones, such as acetone.

Such surfactants include, but are not limited to, lipophilic surfactants, particularly those having an HLB value of 3 to 8, examples of which are span-type surfactants such as span20, 40, 60 or 80, sugar ester surfactants such as glucose esters of C12 to C18 acids; propylene glycol esters of C12-C18 acids; C12-C18 glycerol (glycerol) esters; to co-operate in combination.

The above lipophilic surfactant may be used in combination with 0.1-5 wt% of hydrophilic surfactant, such as TWEEN 80.

Laccase should be added into the aqueous phase before the formation of the water-in-oil emulsion, and should not be added into the oil phase before or after the formation of the water-in-oil emulsion, otherwise the performance of the type I and type V collagen implants is reduced or eliminated.

The volume ratio of the oil phase to the water phase is 2.5 to 10, preferably 3 to 6.

The aqueous solution should be mixed with the oily solution within 30 minutes, preferably within 10 minutes, and most preferably within 5 minutes after the completion of the preparation of the aqueous solution.

Preferably, in the process of filtering the emulsion, particles with the particle size of more than 25 μm and less than 5 μm are filtered, and particles with the particle size of between 5 μm and 25 μm are reserved; most preferably, particles having a particle size of greater than 25 μm and less than 10 μm or particles having a particle size of greater than 15 μm and less than 5 μm are filtered off, particles having a particle size of between 10 μm and 25 μm are retained, or particles having a particle size of between 5 μm and 15 μm are retained.

The low temperature freezing method includes a freeze-drying method and a spray freeze-drying method.

The process of the invention adopts the enzymatic method to crosslink the collagen, and has great advantages compared with the chemical crosslinking process and the physical crosslinking process:

mild reaction conditions, no by-products, high specificity, high catalytic efficiency and yield.

In addition, the mild reaction conditions reduce the loss of collagen during processing.

Examples

The following non-alternative examples, namely methods of making implants, further describe preferred embodiments within the scope of the present invention. Many variations of these embodiments are possible within the scope of the invention.

Example 1

(1) 3.5% of surfactant at a preparation temperature of 4 ℃): ethyl acetate solution of SPAN 80 to obtain oil phase;

(2) Preparing an aqueous solution containing 4% of type I collagen, 1% of type V collagen, 6% of maltitol and laccase at a temperature of 4 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I and type V collagen is 8U/g, and obtaining an aqueous phase;

(3) Mixing said aqueous solution with said oil phase within 15 to 30 minutes after said preparation of said aqueous solution, and vigorously stirring for 30 minutes to form a water-in-oil emulsion, maintaining the temperature at 4 ℃ to 0 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 6;

(4) Filtering out particles with the particle size of more than 25 mu m and less than 0.5 mu m in the emulsion by using a filter membrane after the collagen is basically crosslinked, and retaining the particles with the particle size of between 0.5 mu m and 25 mu m;

(5) Washing the above retained particles with 10-30% ethanol water solution, lyophilizing at-60deg.C to-80deg.C in vacuum freeze dryer for 48 hr, and removing solvent and water.

15 batches were prepared in the same manner.

Example 2

(1) 5% of surfactant at preparation temperature 0 ℃): the solution of the oleic acid glucose ester in diethyl ether is used for obtaining an oil phase;

(2) Preparing an aqueous solution containing 3% of type I collagen, 0.5% of type V collagen, 5% of sorbitol and laccase at a temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I and type V collagen is 5U/g, and obtaining an aqueous phase;

(3) Mixing said aqueous solution with said oil phase within 5 to 10 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 60 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 24 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 5;

(4) Filtering out particles with the particle size of more than 5 mu m and less than 0.5 mu m in the emulsion by using a filter membrane after the collagen is basically crosslinked, and retaining the particles with the particle size of between 0.5 mu m and 5 mu m;

15 batches were prepared in the same manner.

Example 3

(1) 4% of surfactant at preparation temperature 0 ℃): a solution of C14 acid (myristic acid) glyceride in acetone to obtain an oil phase;

(2) Preparing an aqueous solution containing 10% of type I collagen, 2% of type V collagen, 6% of mannitol and laccase at a temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I and type V collagen is 6U/g, and obtaining an aqueous phase;

(3) Mixing said aqueous solution with said oil phase within 5 to 15 minutes after said preparation of said aqueous solution is completed, and vigorously stirring for 30 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 8;

(4) Filtering out particles with the particle size of more than 25 mu m and less than 5 mu m in the emulsion by using a filter membrane after the collagen is basically crosslinked, and retaining the particles with the particle size of between 5 mu m and 25 mu m;

15 batches were prepared in the same manner.

Example 4

(1) 5% of surfactant at preparation temperature 0 ℃): propylene glycol oleate and 0.1% of isoamyl isovalerate of TWEEN 80 to obtain an oil phase;

(2) Preparing an aqueous solution containing 1% of type I collagen, 6% of type V collagen, 8% of erythritol and laccase at a temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I and type V collagen is 6U/g, and obtaining an aqueous phase;

(3) Mixing said aqueous solution with said oil phase within 5 to 25 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 60 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 7;

(4) Filtering out particles with the particle size of more than 15 mu m and less than 5 mu m in the emulsion by using a filter membrane after the collagen is basically crosslinked, and retaining the particles with the particle size of between 5 mu m and 15 mu m;

15 batches were prepared in the same manner.

Example 5

(1) 3% of surfactant at preparation temperature of 0 ℃): butyl acetate solution of SPAN20 is used for obtaining an oil phase;

(2) Preparing an aqueous solution containing 2% of type I collagen, 6% of type V collagen, 8% of lactitol and laccase at a temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I and type V collagen is 5U/g, and obtaining an aqueous phase;

(3) Mixing said aqueous solution with said oil phase within 15 to 30 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 30 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 9;

15 batches were prepared in the same manner.

Example 6

(1) 4% of surfactant at preparation temperature 0 ℃): a solution of sucrose ester of soft ester acid (C16 acid) and methyl acetate of TWEEN 80 of 0.1% to obtain an oil phase;

(2) Preparing an aqueous solution containing 6% of type I collagen, 2% of type V collagen, 5% of xylitol and laccase at the temperature of 1 ℃, wherein the ratio of the dosage of the laccase to the total dosage of the type I and type V collagen is 10U/g, and obtaining an aqueous phase;

(3) Mixing said aqueous solution with said oil phase within 5 to 20 minutes after completion of said preparation of said aqueous solution, and vigorously stirring for 60 minutes to form a water-in-oil emulsion, maintaining the temperature at 0 ℃ to-4 ℃ for 48 hours to substantially crosslink collagen, said oil phase and said water phase having a volume ratio of 5;

15 batches were prepared in the same manner.

Example 7

(4) Filtering out particles with the particle diameter of more than 25 mu m and less than 10 mu m in the emulsion by using a filter membrane after the collagen is basically crosslinked, and retaining the particles with the particle diameter of between 10 mu m and 25 mu m;

15 batches were prepared in the same manner.

Comparative examples 1 to 7-1

The same applies to the respective examples, except that particles having a particle size of greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which is the same as the respective examples) and less than 0.5 μm or 5 μm or 10 μm or 15 μm (which is the same as the respective examples) are not filtered out.

Comparative examples 1 to 7-2

The same applies to the respective examples, except that particles having a particle diameter of greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which are the same as those of the respective examples) are not filtered out.

Comparative examples 1 to 7-3

The same applies to the respective examples, except that particles having a particle diameter of less than 0.5 μm or 5 μm or 10 μm or 15 μm (which is the same as that of the respective examples) are not filtered out.

Comparative examples 1 to 7-4

The same applies to the respective examples, except that the laccase is not added (directly) to the aqueous phase, but to the oily phase.

Comparative examples 1 to 7-5

The examples were the same as the examples, except that no surfactant was added and no particles having a particle size of greater than 25 μm or 5 μm or 10 μm or 15 μm or 20 μm (which was the same as the examples) and less than 0.5 μm or 5 μm or 10 μm or 15 μm (which was the same as the examples) were filtered.

Test example 1 through needle performance test

Principle of:

the better the needle performance, the less time is required for the same amount of suspension to pass through the same needle under the same conditions.

The method comprises the following steps:

the same weight (35 mg, total weight of type I and type V collagens) of the above examples, control and commercial collagen (35 mg, botai: french, and lyophilization for 48 hours at-60- -80℃in a vacuum lyophilization machine was used as a reference) to prepare a lyophilized preparation by removing solvent water, adding the same amount (5 ml) of physiological saline for injection to reconstitute (reconstitute) into a suspension in the same manner (as in the same shaking method) for the same time, loading the same amount of suspension into the same syringe (needle is unchanged, and drying for reuse after washing), pushing the syringe with the same constant pressure, and measuring the required time. Finally, the ratio of the time measured by the above examples and the control example to the time measured by the commercial product is calculated, and the needle passing performance of the above examples and the control example is measured by the ratio, the smaller the ratio is, the stronger the needle passing performance is, and the larger the ratio is, the weaker the needle passing performance is.

The test results are shown in tables 1 to 7.

Test example 2

In vitro degradation time test

The same weight of collagen (35 mg, total weight of type I and type V collagen) as the above example, comparative example and commercially available collagen (35 mg, trade name: botai: french, and lyophilized preparation prepared by removing solvent water by lyophilization at-60- -80℃for 48 hours in a vacuum freeze-dryer as a reference) was taken, and a suspension was reconstituted in the same manner for the same time by adding the same amount (100 ml, concentration 0.035%) of physiological saline for medical injection (pH 7.0). Adding the same amount of collagenase (Bacillus cereus protease, purified and separated from Bacillus cereus) to the suspension, mixing the above collagen protease and collagen at a weight ratio of 4U/mg, and placing in a constant temperature and humidity environment with a temperature of 37deg.C and a relative humidity of 70%, sampling at 0hr, 1 hr, 3 hr, 5 hr, 7 hr, 9 hr, 11 hr, 13 hr, 15 hr, 17 hr, … … until the absorbance value A is substantially stable (fluctuation range is less than 2%), wherein the average value of the stable absorbance value A is expressed as A ₀ 。

After sampling, enzyme is immediately deactivated for 10min at 90 ℃, the temperature is reduced to room temperature, and the centrifugation is carried out for 10min at 4000 r/min, so that clear hydrolysate is obtained. Taking 5mL of the hydrolysate sample solution, adding 5mL of 15% (W/W) trichloroacetic acid (TCA) aqueous solution, uniformly mixing, standing for 10min, centrifuging for 10min at 4 000r/min, diluting the supernatant to a solution with the concentration of 1-10 mg/mL, taking 1mL of the diluent, adding 4mL of biuret reagent, uniformly mixing, and standing for 30min. Absorbance value a was measured at wavelength 540 nm. The concentration C (mg/mL) of polypeptide produced by degradation can be calculated according to a linear regression equation (see document 1: meat industry. 2011 (11), general phase 367, pages 21-24, liu Lili, yang Xieli, kinetics study of collagenase enzymatic hydrolysis of bovine bone collagen).

The absorbance value A (or the concentration C of the polypeptide produced by the degradation) reflects the amount of collagen that has been degraded in the sample solution ₀ -A (or C) ₀ -C，C ₀ Represents the average value A after the absorbance value A is basically stable ₀ The degradation is determinedThe amount of collagen initially reacted, which is the concentration of the polypeptide produced by complete degradation of collagen), reflects the amount of collagen remaining in the sample solution, and since the degradation of protease is characterized by the Emi equation as a first order reaction at lower concentrations (see document 1 above), ln (A) ₀ -A) (or ln (C) ₀ -C)) is plotted against time T, and the degradation slope k is determined by the slope k and the formula T _0.99 Complete degradation of collagen (99% degradation or residual 1%, t=ln (C) ₀ ＇/ C＇)/k, _t C＇/ C ₀ ' 0.01, wherein C ₀ 'and C' respectively represent the amount of initial collagen and the amount of residual collagen) time T _0.99 。

Finally, the time T for complete degradation of the collagen measured in the above examples and comparative examples is calculated _0.99 Time T of complete degradation of collagen measured with commercially available collagen _0.99 The ratio (in terms of its average value) is measured as the relative in vivo retention performance of the above examples and comparative examples, the greater the ratio, the greater the in vivo retention (long-acting) performance and the smaller the ratio, the weaker the in vivo retention (long-acting) performance. The smaller the fluctuation of the ratio, the smaller the change of the in-vivo persistence (long-acting) performance of the collagen; the greater the fluctuation of this ratio, the greater the change in the in vivo persistence (long-acting) properties of collagen.

The test results are shown in tables 1 to 7.

TABLE 1

Example 1

Comparative example 1-1

Comparative examples 1 to 2

Comparative examples 1 to 3

Comparative examples 1 to 4

Comparative examples 1 to 5

Needle passing performance

0.26

0.48

0.56

0.24

-

Degradation time ratio

4.5±1.1

6.5±3.9

8.1±5.3

3.7±2.4

1.3±0.3

10.8±7.2

TABLE 2

Example 2

Comparative example 2-1

Comparative example 2-2

Comparative examples 2 to 3

Comparative examples 2 to 4

Comparative examples 2 to 5

Needle passing performance

0.13

0.32

0.39

0.16

-

Degradation time ratio

2.3±0.4

4.9±2.3

6.5±3.8

1.8±0.6

1.1±0.1

8.7±5.6

TABLE 3 Table 3

Example 3

Comparative example 3-1

Comparative example 3-2

Comparative examples 3 to 3

Comparative examples 3 to 4

Comparative examples 3 to 5

Needle passing performance

0.28

0.44

0.52

0.31

-

Degradation time ratio

5.8±1.5

7.8±4.3

8.9±6.2

4.2±2.7

1.4±0.3

11.4±7.8

TABLE 4 Table 4

Example 4

Comparative example 4-1

Comparative example 4-2

Comparative examples 4 to 3

Comparative examples 4 to 4

Comparative examples 4 to 5

Needle passing performance

0.20

0.37

0.44

0.22

-

Degradation time ratio

3.6±0.8

5.8±3.1

6.9±4.8

3.1±2.0

1.3±0.2

10.5±7.3

TABLE 5

Example 5

Comparative example 5-1

Comparative example 5-2

Comparative examples 5 to 3

Comparative examples 5 to 4

Comparative examples 5 to 5

Needle passing performance

0.30

0.47

0.56

0.28

-

Degradation time ratio

5.3±1.3

7.9±5.2

9.8±7.1

4.1±2.3

1.4±0.3

12.2±9.1

TABLE 6

Example 6

Comparative example 6-1

Comparative example 6-2

Comparative examples 6 to 3

Comparative examples 6 to 4

Comparative examples 6 to 5

Needle passing performance

0.27

0.42

0.56

0.29

-

Degradation time ratio

5.6±1.4

7.9±4.8

9.3±5.7

4.3±2.8

1.5±0.3

12.5±8.9

TABLE 7

Example 7

Comparative example 7-1

Comparative example 7-2

Comparative example 7-3

Comparative examples 7 to 4

Comparative examples 7 to 5

Needle passing performance

0.32

0.57

0.69

0.35

-

Degradation time ratio

7.2±1.6

9.8±7.0

11.9±8.8

5.8±3.2

1.7±0.5

13.9±10.2

The results show that:

1) The examples have a slower rate of enzymatic hydrolysis than the commercial products, significantly longer in vivo retention (long-lasting) performance;

2) The examples have better in vivo retention (long-acting) performance than the control examples without surfactant, which have extremely high in vivo retention (long-acting) performance variability;

3) The examples do not (directly) add to the aqueous phase, but the control added to the oil phase has better in vivo retention (long-lasting) properties, which are very weak;

4) The control examples, which had less filtering of larger and smaller particles, had better in vivo retention (long-term) performance and less volatility, which had very much fluctuation in vivo retention (long-term) performance;

5) The control examples, which were less prone to larger particle removal and which were only prone to smaller particle removal, had better in vivo retention (long-term) performance and less volatility;

6) The comparative example, which is less filtered of smaller particles and only filtered of larger particles, has better in vivo retention (long-acting) performance, longer in vivo retention (long-acting) performance, less volatility, and the comparative example has shorter in vivo retention (long-acting) performance and larger variability;

7) The examples have better needle penetration performance than commercial products, comparative examples that do not filter larger and smaller particles, and comparative examples that do not filter larger particles but only smaller particles; the examples have substantially the same needle passing performance as the control with only larger particles without smaller particles.

Claims

1. A preparation method of a microparticle type I and type V collagen composite implant with long-acting and small fluctuation of the effective period and good needle penetration comprises the following steps: