CN106924809B

CN106924809B - I type collagen and liquid submucosal filling agent

Info

Publication number: CN106924809B
Application number: CN201710167939.0A
Authority: CN
Inventors: 张慧敏; 富勇; 姜智旭
Original assignee: Beijing Hotwire Medical Tech Development Co ltd
Current assignee: Beijing Hotwire Medical Tech Development Co ltd
Priority date: 2017-03-20
Filing date: 2017-03-20
Publication date: 2020-06-23
Anticipated expiration: 2037-03-20
Also published as: CN106924809A

Abstract

The invention discloses type I collagen and a liquid submucosal filling agent, wherein the collagen has a complete natural triple helix structure, and the content of hydroxyproline is more than 11 percent. This type I collagen and by the type I collagen powder of type I collagen preparation, specially adapted preparation liquid submucosal filler, the swelling agent of focus and the prosthetic materials of operation in-process wound in scope submucosal resection or scope submucosal stripping operation can be used as to the filler under the obtained liquid mucosa, and the support time length is reasonable, can be absorbed by the tissue completely, has hemostasis restoration ability function concurrently simultaneously, and the result of application is very good.

Description

I type collagen and liquid submucosal filling agent

Technical Field

The invention relates to the field of biological materials, in particular to type I collagen and a liquid submucosal filling agent prepared from the type I collagen.

Background

At present, the detection rate of precancerous lesion and early cancer gradually increases with the popularization of diagnostic endoscopy. Endoscopic Submucosa Dissection (ESD) or endoscopic submucosa resection (EMR) gradually becomes a standard minimally invasive treatment means for resection of precancerous lesions and precancerous lesions of the digestive tract, and can completely and integrally resect the lesions, thereby greatly reducing the risk of tumor recurrence.

ESD surgery also presents a significant risk and may present complications, including digestive tract perforation and bleeding. In order to make the perforation of the patient safer and more convenient, most researchers at home and abroad currently confirm that the occurrence of the complications can be reduced by performing submucosal injection before surgical resection.

The submucosal injection currently studied includes normal saline, hypertonic saline, glucose, glycerol fructose, sodium hyaluronate with different concentrations, hydroxymethyl cellulose, fibrinogen and the like. However, these submucosal injections still have some disadvantages in clinical applications. For example, after the saline injection, the liquid spreads rapidly to the surrounding tissues and the time for maintaining the swelling of the diseased mucous membrane is short, and most of the local swelling shape formed by the saline injection has partially disappeared when the excision operation has not been completed. Other injections, such as hypertonic saline, have tissue damaging effects to varying degrees.

Therefore, the invention is especially provided.

Disclosure of Invention

The invention aims to provide a novel type I collagen and develop a novel functional structure of the type I collagen.

Another objective of the invention is to provide a new application of the novel type I collagen in preparing a submucosal injection.

It is a third object of the present invention to provide a novel submucosal injection to overcome the problems associated with the use of the existing submucosal injections.

The invention realizes the aim through the following technical scheme:

the invention provides a type I collagen which has a complete natural triple-helix structure, wherein the content of hydroxyproline is more than 11 percent (the hydroxyproline in the type I collagen accounts for about 9-13 percent of the collagen, see the 6 th part of tissue engineering medical products in the Chinese people's republic of China medical and pharmaceutical industry standard: type I collagen). Researches find that the collagen has good thermal stability, lasting space supporting effect, moderate time length of the effect of swelling the focus, no need of taking out the collagen in the later period, and timely hemostasis effect on the oozing blood at the wound of the operation, thus being beneficial to the rapid repair of the wound.

Based on these characteristics, the invention provides the application of the type I collagen in preparing the liquid submucosal filling agent.

The liquid submucosa filling agent is injected into the pathological submucosa to form a submucosa liquid cushion layer between the pathological change and the intrinsic muscular layer to support the pathological mucosa, prevent perforation when the pathological mucosa is removed, play a role in stopping bleeding and promote the healing of the removed surface. The liquid submucosa filling agent can be applied to the mucosa of the digestive organs where lesions occur.

Although there are many biomaterials with collagen as matrix in the market, such as collagen sponge, absorbable collagen suture, medical collagen film, collagen plaster, collagen implant (Shuangmei), medical collagen filler (Fumeida), etc., for ESD or EMR operation, it is required that the submucosal liquid pad can continuously support during the operation, and can be automatically absorbed by tissues after the operation is completed without being taken out to avoid secondary damage to patients, so the existing collagen matrix biomaterials can not be applied.

The invention also provides I type collagen powder which is most suitable for preparing the liquid submucosal filling agent, and the preparation method specifically comprises the following steps:

(1) the type I collagen of claim 1 added to a neutral buffer solution, stirred uniformly, left to stand and soaked;

(2) adding formaldehyde, and stirring at 35-39 ℃ to perform a crosslinking reaction;

(3) washing a reaction product by using a neutral buffer solution, and then washing by using deionized water;

(4) freeze drying, and crushing to obtain particles with the particle size of 200-300 microns to obtain the collagen powder.

As a preferred scheme, the preparation method of the type I collagen powder specifically comprises the following steps:

(1) adding the type I collagen into a neutral buffer solution, uniformly stirring, standing and soaking;

The whole system of the solution can be in a uniform and stable pH environment by adding neutral buffer solution and standing and soaking, and when a meta-acid or meta-alkali substance is added, a certain buffering effect can be exerted on the pH value of the solution, so that the pH value cannot cause great change.

Because collagen is degraded when stored in liquid for a certain period of time, the triple helix structure of the collagen is damaged, and the practical application efficacy of the collagen is weakened or lost. Therefore, formaldehyde is added for crosslinking, so that the degradation time can be prolonged. The filler prepared from the non-crosslinked collagen can be used in principle, but the non-crosslinked collagen is degraded in a liquid more quickly than the crosslinked collagen, so that the effective period of the product is short; while cross-linking may have some effect on the viscosity of the final liquid filler. The purpose of the neutral buffer washing and the deionized water washing is to remove formaldehyde remaining without participating in the reaction.

Because collagen belongs to a biological material which is sensitive to temperature and can be denatured when the temperature exceeds a certain temperature, the material is crushed under the condition of liquid nitrogen at present; in addition, even under the condition of liquid nitrogen, the toughness of the steel is still very high. Therefore, the average particle size of the particles cannot be reduced to 150 μm or less by merely grinding the particles under the liquid nitrogen condition. Therefore, a high-pressure homogenization process described later is also required.

The crosslinking temperature of the type I collagen is lower than the denaturation temperature of the type I collagen, and the type I collagen is crushed at the temperature of below 196 ℃ below zero after freeze drying, so that the specific natural triple helical structure in the type I collagen is reserved, the triple helical structure is prevented from being dispersed to generate denaturation due to overhigh temperature, and a better using effect is achieved.

As a preferable scheme, the type I collagen powder is prepared by mixing 0.01mol/L disodium hydrogen phosphate solution and 5-6 pH MES solution until the pH is 7 and diluting the mixture by 20 times with deionized water. Since the disodium hydrogen phosphate solution is too concentrated or too low in pH, which results in dissolution of collagen, a 0.01mol/L disodium hydrogen phosphate solution and an MES solution having a pH of 5 to 6 were mixed to prepare a buffer.

As a preferred scheme, the volume ratio of the mass of the formaldehyde in the step (2) to the volume of the neutral buffer solution used in the step (1) of the type I collagen powder is 0.08-0.30%.

The product has no strict requirement on the degree of crosslinking, but the crosslinking with proper amount of formaldehyde can improve the viscosity of the final liquid filling agent and prolong the effective period of the product.

As a preferred scheme, in the step (2), the stirring speed is 180-300 r/m, and the reaction time is 3-4 h. Under the reaction condition, the crosslinking can be more uniform, and a better crosslinking effect is achieved.

As a preferable scheme, the I type collagen powder is lyophilized in the step (4) according to the following steps:

a. pre-freezing: pre-freezing for 60min at-40 ℃, then pre-freezing for 150-180 min at-20 ℃, and keeping the temperature of a freezer of a freeze dryer at-60 to-50 ℃;

b. and (3) drying: drying at-10 deg.C for 7h, drying at 0 deg.C for 7h, drying at 5 deg.C for 8h, drying at 10 deg.C for 8h, drying at 1510 deg.C for 8h, and drying at 20 deg.C for 20 h;

the environmental vacuum degree in the pre-freezing and drying stage is 200-350 mTorr.

The pre-freezing and the drying are arranged, so that the moisture content in the sample to be freeze-dried can be reduced to below 10 percent, and the next step of freeze grinding is facilitated.

As a preferable scheme, the type I collagen powder is crushed in the step (4) by adopting freeze grinding, the ambient temperature is below 196 ℃, and the crushing time is 10-12 min.

At very low temperatures, the sample becomes brittle, is easier to grind and meets the required particle size requirements; meanwhile, the grinding is carried out at the temperature, so that the triple-helix structure of the collagen is not damaged.

The invention also provides a liquid submucosa filling agent, which is prepared by suspending the type I collagen or any type I collagen powder in phosphate buffer solution or physiological saline; the average particle size of collagen in the suspension is 50 to 150 μm.

The above particle size requirements are required to be achieved because clinical injections require passage through the associated mucosectomy catheter.

As a preferred scheme, when the I-type collagen powder is used, the I-type collagen powder is added into a phosphate solution or physiological saline, and the mixture is homogenized under the conditions that the temperature is below 0 ℃ and the pressure is 800-1000 bar until the particle size of collagen is reduced to 50-150 mu m, so that the liquid submucosa filler is obtained.

The temperature is set below 0 ℃ to ensure that the protein is not denatured in the preparation process; the high pressure may reduce the average homogeneous particle size of the collagen particles.

As a preferred scheme, the homogenization of the liquid submucosa filling agent is repeated for 5-10 times.

Repeated homogenization can homogenize the average particle size of the collagen particles and better disperse the collagen particles uniformly in the solution.

The invention has the following beneficial effects:

the invention uses the type I collagen which only contains a natural triple-helix structure and has the hydroxyproline content of more than 11 percent as a raw material to prepare the liquid submucosa filling agent, can be used as a swelling agent of focuses in endoscopic submucosa resection or endoscopic submucosa dissection, is maintained for a long time enough to ensure that the operation is smoothly implemented without multiple injections, can not damage surrounding tissues, and effectively reduces complications such as bleeding, perforation and the like; the product can also be used as a repair material for wounds in the operation process, can not bleed after the operation, can be degraded and absorbed within 30 days, and does not worry about the adverse effect on tissues.

Detailed Description

The following detailed description further details the invention in order to provide those skilled in the art with a better understanding of the inventive concepts.

Example one

Adding MES solution with pH 6 into 0.01mol/L disodium hydrogen phosphate solution until the pH of the mixture is 7, and diluting the mixture to be neutral with deionized water by 20 times to prepare neutral buffer solution.

20g of type I collagen (the content of hydroxyproline is more than 11%) with the purity of more than 99% and a natural triple-helix structure are put into 600ml of neutral buffer solution, stirred for 5-10 min until the mixture is uniform, and then are kept stand and soaked for 1.5 h.

Adding 1.25ml of 38.5% formaldehyde solution into neutral buffer solution, stirring for 7min to mix uniformly, and then placing in a 37 ℃ constant temperature water bath kettle to stir at the speed of 200 rpm for reaction and crosslinking for 3 h.

And after the reaction is finished, taking out the reaction product, washing the reaction product for 30min by using buffer solution, and then washing the reaction product for 1h by using deionized water.

Freeze-drying the cleaned collagen, pre-freezing for 1h at-40 ℃, and then pre-freezing for 3h at-20 ℃, wherein the temperature of a freezer of the freeze dryer is kept between-60 ℃ and-50 ℃.

The freeze drying process comprises drying at-10 deg.C, drying at 0 deg.C for 7 hr, drying at 5 deg.C for 8 hr, drying at 10 deg.C for 8 hr, drying at 15 deg.C for 8 hr, and drying at 20 deg.C for 20 hr. The water content of the dried material is about 6 percent. The ambient vacuum during pre-freezing and drying was 200 mtorr.

And (3) freezing, grinding and crushing the dried material at the temperature of below 196 ℃ below zero for 10min, and screening by using a screen to obtain the 0.08% formaldehyde cross-linked type I collagen powder with the particle size of 200-300 microns.

Example two

And (3) taking 20g of type I collagen with a purity of more than 99% and a natural triple-helical structure, putting the type I collagen in 600ml of buffer solution, stirring for 5-10 min until the type I collagen is uniform, standing and soaking for 1.5 h.

Adding 1.56ml of 38.5% formaldehyde solution into the buffer solution, stirring for 10min to mix uniformly, and then placing the mixture into a 37 ℃ constant temperature water bath kettle to stir at the speed of 220 rpm for reaction and crosslinking for 3.5 h.

After the completion of the collagen extraction, the collagen was washed with buffer solution for 40min and then with deionized water for 1.5 h.

Freeze drying collagen, pre-freezing at-40 deg.c for 1 hr, and pre-freezing at-20 deg.c for 3 hr while maintaining the temperature in the freezer of the freeze drier at-60 deg.c to-50 deg.c.

The freeze drying process comprises drying at-10 deg.C, drying at 0 deg.C for 7 hr, drying at 5 deg.C for 8 hr, drying at 10 deg.C for 8 hr, drying at 15 deg.C for 8 hr, and drying at 20 deg.C for 20 hr. The water content of the dried material is about 6 percent. The ambient vacuum during pre-freezing and drying was 300 mtorr.

And (3) freezing, grinding and crushing the dried material at the temperature of below 196 ℃ below zero for 11min, and screening by using a screen to obtain the 0.1% formaldehyde cross-linked type I collagen powder with the particle size of 200-300 microns.

EXAMPLE III

4g of the collagen powder obtained in example one or example two was weighed out, added to 100ml of a phosphate buffer solution having a concentration of 0.5mmol/L and a pH of 7.5, and stirred uniformly.

Then homogenizing the mixed solution at low temperature and high pressure, wherein the homogenizing temperature is below 0 ℃, and the homogenizing pressure is 800 bar. Homogenization was repeated 9 times. The liquid submucosa filling agent with the collagen particle size of 50-150 mu m and the collagen concentration of 40mg/ml is obtained.

Example four

5g of the collagen powder obtained in example one or example two was weighed out, added to 100ml of a phosphate buffer solution having a concentration of 0.5mmol/L and a pH of 7.6, and stirred uniformly.

And then homogenizing the mixed solution at low temperature and high pressure, wherein the homogenizing temperature is below 0 ℃, and the homogenizing pressure is 900 bar. Homogenization was repeated 11 times. The liquid submucosa filling agent with the collagen particle size of 50-150 mu m and the collagen concentration of 50mg/ml is obtained.

EXAMPLE five

6g of the collagen powder obtained in example one or example two was weighed out, added to 100ml of a phosphate buffer solution having a concentration of 0.5mmol/L and a pH of 7.8, and stirred uniformly.

And then homogenizing the mixed solution at low temperature and high pressure, wherein the homogenizing temperature is below 0 ℃, and the homogenizing pressure is 900 bar. Homogenization was repeated 13 times. The liquid submucosa filling agent with the collagen particle size of 50-150 mu m and the collagen concentration of 60mg/ml is obtained.

TABLE 1 comparison of filling effect of different sub-mucosal injection products

Referring to table 1, compared with other submucosal injection products such as normal saline, hypertonic saline, glucose, glycerol fructose, sodium hyaluronate with different concentrations, hydroxymethyl cellulose, fibrinogen and the like, the liquid submucosal filling prepared from the type I glue raw material provided by the invention has better effects of stopping bleeding and promoting healing. Meanwhile, because the collagen is a structural protein of the extracellular matrix, the liquid submucosa filling agent prepared from the type I collagen raw material also has biocompatibility, low immunogenicity and biodegradability.

The invention provides a high-purity I-type collagen material with a triple-helix structure, a liquid submucosal filling agent prepared from the I-type collagen material and a preparation method thereof. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the core concepts of the present invention. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A liquid submucosa filling agent is characterized in that the liquid submucosa filling agent is prepared by suspending type I collagen powder in phosphate buffer solution or physiological saline; the average particle size of collagen in the suspension is 50-150 mu m; the collagen has a complete natural triple-helix structure, and the content of hydroxyproline is more than 11 percent;

the preparation method comprises the following steps:

(1) adding the type I collagen into a neutral buffer solution, uniformly stirring, standing and soaking; the neutral buffer solution is prepared by mixing 0.01mol/L disodium hydrogen phosphate solution and MES solution with pH of 5-6 to pH of 7 and diluting the mixture by 20 times with deionized water;

(2) adding formaldehyde, and stirring at 35-39 ℃ to perform a crosslinking reaction; the concentration of the formaldehyde added into the neutral buffer solution used in the step (1) is 0.08-0.30%;

(4) freeze drying, and crushing to obtain particles with the particle size of 200-300 mu m to obtain collagen powder;

(5) adding the type I collagen powder into a phosphate buffer solution or physiological saline, and carrying out high-pressure homogenization at the temperature of below 0 ℃ and under the pressure of 800-1000 bar until the average particle size of the collagen is reduced to 50-150 mu m to obtain the liquid submucosa filler.

2. The liquid submucosa filling agent according to claim 1, wherein the stirring speed in step (2) is 180 to 300 rpm, and the reaction time is 3 to 4 hours.

3. The liquid submucosa bulking agent of claim 1, wherein the lyophilization in step (4) is performed according to the following steps:

b. and (3) drying: drying at-10 deg.C for 7h, drying at 0 deg.C for 7h, drying at 5 deg.C for 8h, drying at 10 deg.C for 8h, drying at 15 deg.C for 8h, and drying at 20 deg.C for 20 h;

4. The liquid submucosa filling agent according to claim 1, wherein the pulverization in step (4) is performed by freeze grinding pulverization, the ambient temperature is below-196 ℃, and the pulverization time is 10-12 min.