CN116492499A - A kind of preparation method of epoxy hyaluronic acid modified acellular dermal matrix, its prepared product and application - Google Patents

Abstract

The invention discloses a preparation method of an epoxy hyaluronic acid modified acellular dermal matrix, which comprises the following steps: hydroxyl on hyaluronic acid reacts with epichlorohydrin, epoxy hyaluronic acid is generated after ring closure, and then the epoxy hyaluronic acid is crosslinked with acellular dermal matrix to prepare the epoxy hyaluronic acid modified acellular dermal matrix. The specificity of collagen in the matrix is synergistic with the specific properties of hyaluronic acid, and when the matrix is contacted with water, microgels can be formed, thus producing a strong "water-locking" effect. The epoxy hyaluronic acid modified decellularized dermal matrix obtained by the invention not only has excellent hydrophilicity and moisture retention, but also has good biocompatibility and excellent wound healing promoting function, is a novel moisture retention biomedical material, and can be widely applied to the field of biological materials.

Description

A preparation method of epoxy hyaluronic acid modified acellular dermal matrix, and its preparation products and applications

technical field

The present invention relates to a preparation method of epoxy hyaluronic acid modified acellular dermal matrix material, also relates to the epoxy hyaluronic acid modified acellular dermal matrix material prepared according to the method and its application, belonging to the field of biomedical materials .

Background technique

Wound healing is a complex process, and the recovery process may be affected by various factors, which may affect the recovery after injury. The traditional concept (dry wound healing) is that exposing the wound to air to allow it to breathe can promote the recovery of wound tissue cells. According to Winter's theory of moist healing, acute superficial wound regeneration (epithelial cells) is twice as fast in a moist environment as in a dry scab wound. When the wound was covered and kept moist, angiogenesis was accelerated, and the inflammatory response of the wound was significantly lower than that of the wound exposed to air. In a humid environment, the recovery speed of the wound tissue will be accelerated, thereby realizing the control of the scab rate, so that the wound can recover in a shorter time (WINTER G D. Formation of the scab and the rate of epithelization of superficial wounds in the skin of the young domestic pig. [J]. Nature, 1962, 193). Moist healing theory therapy can keep the wound surface in a constant temperature state during the recovery process, and prevent the patient's granulation tissue from being affected by external environmental factors during the wound recovery process, and corresponding moist dressings have also emerged as the times require.

Common moisturizing dressings include polyurethane foam and film, synthetic polymer hydrogel, alginate, carboxymethyl cellulose, etc. (Zheng Yunhui, Zhu Qunye. Moisturizing dressings and wound healing[J]. Nursing and Rehabilitation, 2007, 6( 3): 157-159). Polyurethane foam dressings have good air permeability and can prevent excessive evaporation of moisture from the wound, but have almost no absorption properties. Synthetic molecular hydrogels, such as polypropylene amine and polyoxyethylene hydrogels, have a complete three-dimensional structure and contain about 96% water; amorphous hydrogels contain a large amount of water, which can provide moisture to the wound. The above two materials are artificially synthesized materials, and monomer residues inevitably exist during the synthesis process, which affects their biocompatibility. And both are biologically inert substances, lack active ingredients to promote wound healing, and have low biological activity. Natural polysaccharides such as alginate dressing and carboxymethyl cellulose have high hygroscopicity, gelation and hemostatic properties, and have good hydrophilic and moisturizing functions. However, compared with biological dressings (such as acellular dermal matrix), its biological activity is still low and cannot meet the needs of clinical application.

Acellular dermal matrix (ADM) refers to the material obtained by retaining the collagen fiber scaffold and removing the epidermal and dermal cellular components in animal skin. It has excellent mechanical properties and biocompatibility, and can be used as a medical substitute for the dermis, thereby avoiding wound exposure and preventing bacterial invasion, etc. Clinical research and application progress of cellular dermal matrix[J]. Journal of Biomedical Engineering, 2017, 40(1):58-61).

Although collagen, the main component of the acellular dermal matrix, has a certain degree of hydrophilic and moisturizing properties, compared with highly hydrophilic natural polysaccharides (such as sodium alginate, hyaluronic acid, etc.), its hydrophilic and moisturizing properties are still insufficient. In the clinical application of acellular dermal matrix, due to cross-linking, freeze-drying and other treatments, some hydrophilic groups will be blocked, resulting in poor hydrophilic and moisturizing properties. Especially when it is used as a biological dressing, it is prone to problems such as edge warping, fast drying, and hardening. Therefore, how to improve the hydrophilic and moisturizing properties of the acellular dermal matrix is a problem worth studying.

Hyaluronic acid is a straight-chain polymeric acidic mucopolysaccharide composed of D-glucuronic acid and N-acetylglucosamine alternately linked by disaccharide units. It is an important component of cell matrix and various tissues. It has moisturizing, Physiological functions such as lubrication, nutrition, repair and injury prevention, as well as medical functions such as promoting wound healing, promoting angiogenesis, anti-tumor and immune regulation, have been widely used in the fields of medicine and cosmetics. It is not difficult to speculate that if the acellular dermal matrix is immersed in the hyaluronic acid solution, it is expected to improve its hydrophilic and moisturizing properties. In fact, using glycerin, sodium lactate, sodium alginate, hyaluronic acid, etc. as humectants to impregnate the acellular dermal matrix can improve its hydrophilic moisturizing performance in a short time. However, this method has the disadvantages of poor binding ability between the moisturizer and the acellular dermal matrix, and when there is body fluid exudation, the moisturizer is prone to migration and loss, and cannot fundamentally solve the problem of lasting hydrophilic moisturizing. It can be seen that solving the problem of combining the moisturizing agent with the acellular dermal matrix is the key to the above problems.

Using sodium periodate to oxidize polysaccharides can obtain chemically reactive dialdehyde polysaccharides (Yu Guofei, Dan Nianhua. Research progress on oxidized polysaccharides[J]. Biomedical Engineering and Clinic, 2019, 23(1): 105- 110). Then react the oxidized polysaccharide with the acellular dermal matrix to obtain the polysaccharide-modified acellular dermal matrix. For example, by first preparing oxidized hyaluronic acid through this method, and then reacting with acellular dermal matrix, hyaluronic acid molecules can be introduced into ADM, thereby improving its hydrophilic and moisturizing properties. However, as shown in Figure 1, after sodium periodate oxidizes hyaluronic acid, the C-C bond of the adjacent diol structure in the sugar molecule will be broken to form two aldehyde groups, resulting in the breakage of the sodium hyaluronate molecular chain, and the sugar unit is formed from a ring The structure becomes an open-chain structure; at the same time, in the process of oxidation, sometimes the molecular weight decreases. The integrity and high molecular weight of the sugar ring structure are the key factors for the excellent hydrophilic and moisturizing properties of hyaluronic acid. The higher the degree of oxidation, the more serious the damage to the sugar ring and the smaller the molecular weight. This oxidation destroys the integrity of its molecular chain structure, affecting the formation of a "water-locking structure" between hyaluronic acid and water, which in turn leads to its inferior hydrophilic and moisturizing properties. In addition, due to the formation of aldehyde groups after oxidation, the biocompatibility of the product will be more or less adversely affected.

Contents of the invention

The purpose of the present invention is to provide a preparation method of epoxy hyaluronic acid modified acellular dermal matrix, its prepared products and applications. The method of the present invention can not only ensure the integrity of the hyaluronic acid sugar ring but also make it remain It has excellent reactivity, and further, the product obtained by the method of the present invention has excellent hydrophilic moisture retention, biocompatibility and other related properties.

The present invention provides on the one hand:

A preparation method of epoxy hyaluronic acid modified acellular dermal matrix, comprising the following steps:

(1) Preparation of epoxy hyaluronic acid: dissolve hyaluronic acid in a mixed solvent, add a catalyst, add epichlorohydrin to carry out a ring-opening reaction; then in an alkali solution, carry out a ring-closing reaction; then purify and dry to obtain epoxy hyaluronic acid;

(2) Modified cross-linking: the acellular dermal matrix material is modified and cross-linked with the epoxy hyaluronic acid prepared in step (1) to obtain an epoxy hyaluronic acid modified acellular dermal matrix.

In some embodiments of the present invention, in step (1), the mixed solvent is a mixed solvent of an organic solvent and water, and the organic solvent is selected from dimethyl sulfoxide, 1,4-dioxane, N , one or more of N dimethylformamide, diethyl ether or acetonitrile, the volume ratio of the organic solvent to water in the mixed solvent is 1:1˜1:1.5, preferably 1:1.

In some embodiments of the present invention, in step (1), the catalyst is sodium borohydride or tetrabutylammonium bromide.

In some embodiments of the present invention, in step (1), the temperature of the ring-opening reaction is 30-80°C, preferably 60°C-70°C; more preferably 60°C; the temperature of the ring-opening reaction The time is 2 to 8 hours.

In some embodiments of the present invention, in step (1), the alkaline solution is 2-50% aqueous solution or ethanol solution of sodium hydroxide or potassium hydroxide.

In some embodiments of the present invention, in step (1), the temperature of the ring closure reaction is 4-25°C, such as 15°C, 20°C or 25°C; the reaction time is 1-4 hours, such as 2 hours.

In some embodiments of the present invention, in step (1), the purification includes the following steps: adding ethanol to the reacted solution, standing at 0-5°C, preferably at 4°C, for 12- After 48 hours, the precipitate was collected, dissolved in deionized water, put into a dialysis bag, immersed in distilled water for dialysis, and the dialysis product was collected.

In some embodiments of the present invention, the molecular weight cut-off of the dialysis bag is 500-14000 Da, and the dialysis method is: first dialyze for 60-120 minutes, discard the distilled water; then add distilled water and dialyze for 240-360 minutes, discard Distilled water; then dialyze in distilled water for 72 hours, during which the distilled water was changed every 24 hours.

In some embodiments of the present invention, in step (1), the drying adopts a freeze-drying method or a vacuum drying method.

In some embodiments of the present invention, in step (1), based on 100 parts by weight of hyaluronic acid, the added amount of the mixed solvent is 500 to 5000 parts by weight, and the added amount of the catalyst is 5 to 50 parts by weight. parts by weight, the added amount of epichlorohydrin is 20-100 parts by weight, the alkali solution is 50-500 parts by weight, and the added amount of ethanol is 500-2000 parts by weight.

In some embodiments of the present invention, in step (2), the modified crosslinking includes the following steps: weighing 100 parts by weight of the acellular dermal matrix material, adding 100 to 1000 parts by weight of pH 7.0 to 10.5 buffer solution, soak for 10 to 60 minutes; then add 1 to 50 parts by weight of epoxy hyaluronic acid obtained in step (1), and treat for 12 to 72 hours; discard the waste liquid, and add 200 to 1000 parts by weight of hyaluronic acid Wash with water for 10 to 30 minutes; discard the cleaning waste liquid, add 100 to 1000 parts by weight of a buffer solution with a pH of 7.0 to 10.5, add 1 to 10 parts by weight of amino acid or amino acid salt, and treat at 25 to 47 ° C for 30 to 120 min; Discard the waste liquid, add 200-1000 parts by weight of pure water to wash for 10-30 minutes; discard the waste liquid, add 200-1000 parts by weight of pure water to wash for 10-30 minutes; discard the waste liquid, add 5-10 parts by weight of Ammonium chloride or lactic acid, react for 30-60 minutes; discard the waste liquid, add 200-1000 parts by weight of water for injection at 20-40 °C, wash for 60-120 min; discard the waste liquid, add 200-1000 parts by weight of water at 20-40 °C injection water, and wash for 60-120 minutes; obtain epoxy hyaluronic acid modified acellular dermal matrix.

In some embodiments of the present invention, the acellular dermal matrix (acellular.dermal matrix, ADM) is selected from acellular porcine dermal matrix (pADM), acellular fetal bovine dermal matrix (fcADM), acellular goat dermal matrix ( gADM) and decellularized bovine dermal matrix (bADM); the buffer solution is a phosphate buffer solution, sodium carbonate-sodium bicarbonate buffer solution or sodium carbonate-sodium hydroxide buffer solution; the amino acid glutamic acid or aspartic acid, and the amino acid salt is sodium glutamate or sodium aspartate.

Another aspect of the present invention provides the epoxy hyaluronic acid modified acellular dermal matrix prepared by the preparation method of the present invention.

In some embodiments of the present invention, the shrinkage temperature of the epoxy-hyaluronic acid-modified acellular dermal matrix is 62-78°C, the contact angle is 10-50°, the moisture absorption rate is 400-600%, and the water retention rate is 150-300%, the water loss rate is 20-40%, the equilibrium adsorption capacity is 250-400mg/g, and the cytotoxicity of the extract is 0-1.

Another aspect of the present invention provides the use of the epoxy-hyaluronic acid-modified acellular dermal matrix of the present invention in the preparation of moisturizing dressings.

Another aspect of the present invention provides epoxy hyaluronic acid prepared in step (1) of the preparation method of the present invention.

Beneficial effect:

The method of the invention prepares a special modified cross-linking agent-epoxy hyaluronic acid (EHA). Epoxy hyaluronic acid with different epoxy values can be prepared by selecting the molecular weight of hyaluronic acid and controlling reaction conditions (such as material ratio, temperature, time, etc.) according to product requirements.

The specificity of collagen in the epoxy-hyaluronic acid-modified acellular dermal matrix obtained by the method of the present invention and the specificity of hyaluronic acid can produce a synergistic effect, and when the matrix is in contact with water, it can form a microgel, thereby producing a strong "Water lock" effect. The epoxy hyaluronic acid modified acellular dermal matrix obtained in the present invention not only has excellent hydrophilicity and moisture retention, but also has good biocompatibility and excellent function of promoting wound healing, without skin sensitization, without Pyrogen, the cytotoxicity of its extract is 0-1; the stability is high; the controllability is good, and the expandability is good. It is a new type of moisturizing biomedical material and can be widely used in the field of biomaterials.

Description of drawings

Figure 1 Schematic diagram of the reaction of sodium periodate to oxidize hyaluronic acid.

Fig. 2 Schematic diagram of the preparation principle of epoxy hyaluronic acid.

Figure 3 is a schematic diagram of the appearance of hyaluronic acid (a) and epoxy hyaluronic acid (b).

Figure 4 is a diagram of the water-locking structure of hyaluronic acid coils.

Fig. 5 Schematic diagram of microgel formed by epoxy hyaluronic acid grafted with ADM.

Fig. 6 is a schematic diagram of the microstructure after cross-linking of epoxy hyaluronic acid and ADM in different amounts.

Figure 7 Reaction of epoxy hyaluronic acid with ADM.

Figure 8 Infrared spectra of hyaluronic acid and epoxy hyaluronic acid.

Figure 9 NMR spectra of epoxy hyaluronic acid and hyaluronic acid.

Detailed ways

Unless otherwise stated in context, terms in the present invention have the following meanings.

The terms "having", "comprising" and "comprising" should be interpreted as open-ended, indicating the presence of the listed elements but not excluding the existence, presence or addition of any other unlisted element or elements.

All ranges recited herein include those endpoints recited as a range between two values unless otherwise indicated to the contrary. All values recited herein, whether indicated or not, include the degree of experimental, technical, and instrumental error expected for the given technique used in measuring the value.

The term "epoxy hyaluronic acid (EHA)" refers to hyaluronic acid derivatives formed by introducing epoxy groups into hyaluronic acid molecules. The synthetic method of epoxy hyaluronic acid of the present invention is shown in Figure 2. The epoxy hyaluronic acid synthesized by the present invention is outside the six-membered sugar ring. The synthesis method of the present invention does not destroy the sugar ring of hyaluronic acid, so it can obtain excellent chemical reactivity without destroying the structural integrity of the sugar ring, while maintaining excellent biocompatibility and other related properties. The present invention can prepare epoxy hyaluronic acid with different epoxy values by selecting the molecular weight of hyaluronic acid and controlling reaction conditions (such as material ratio, temperature, time, etc.) according to product requirements.

The term "aceiiular dermal matrix (ADM)" refers to the complete removal of the epidermis and cellular components in human or animal skin by logistics, chemical and other methods, leaving only the extracellular matrix containing collagen framework in the dermis. The acellular dermal matrix (ADM) in the present invention may be, for example, acellular porcine dermal matrix (p-ADM), acellular fetal bovine dermal matrix, acellular goat dermal matrix, and acellular bovine dermal matrix.

The term "epoxy hyaluronic acid modified acellular dermal matrix (EHA-ADM)" refers to a product obtained by modifying and crosslinking epoxy hyaluronic acid and acellular dermal matrix. The epoxy hyaluronic acid modified acellular dermal matrix in the present invention can be such as epoxy hyaluronic acid modified acellular porcine dermal matrix (pADM), epoxy hyaluronic acid modified acellular fetal bovine dermal matrix, epoxy hyaluronic acid modified acellular Hyaluronic acid modified acellular goat dermal matrix and epoxy hyaluronic acid modified acellular bovine dermal matrix, etc.

The epoxy group on the epoxy hyaluronic acid of the present invention can react with active groups such as amino groups on the acellular dermal matrix (collagen) to form a firm covalent bond, so that the hyaluronic acid molecule is firmly anchored in the ADM superior. Since the sugar ring structure of hyaluronic acid molecules is complete and the molecular weight is relatively large, it still maintains excellent hydrophilic and moisturizing properties; at the same time, the main chain structure is still composed of hyaluronic acid sugar units, thus retaining its excellent biocompatibility; in addition , after the epoxy group reacts with collagen, the cytotoxicity of the product is much lower than that of aldehyde crosslinking. When the acellular dermal matrix modified by epoxy hyaluronic acid contacts with water, the hyaluronic acid grafted on the surface of collagen fibers exerts a strong hydrophilic and moisturizing effect, firmly "locking" water molecules on its sugar chains, Together with the hydrophilic and moisturizing properties of collagen itself, it builds a moisture-absorbing and moisturizing microenvironment for the defect site, and promotes the healing and repair of wounds at the defect site under the synergy of the two excellent biocompatibility and biological activities (hemostasis, healing promotion, etc.) .

As shown in Figure 4, the polar groups contained in hyaluronic acid molecules, such as carboxyl and hydroxyl groups, can form hydrogen bonds with water to bind a large amount of water. In aqueous solution, its molecules form arbitrary coiled "coils" and fill all spaces, maximally binding with water. The water-holding capacity of a single hyaluronic acid is 2×10 ³ -6×10 ³ ml/g, which can bind 1000 times its weight in water. For example, the vitreous body of the animal eye is composed of hyaluronic acid and collagen fibers, in which the collagen acts as a solid support, the hyaluronic acid is embedded in it, and is filled with a large amount of water, thus forming a gel, which can regulate the water in the vitreous body.

As shown in Figure 5, when the epoxy hyaluronic acid is grafted onto the acellular dermal matrix (collagen fibers), the hyaluronic acid polymer is anchored on the collagen fibers, and when it contacts with water molecules, due to the grafting The integrity of the hyaluronic acid molecule is similar to that of the natural hyaluronic acid molecule, therefore, it can form any curly "coil" and fill all the spaces like the natural hyaluronic acid molecule, and combine with water to the greatest extent. The structure it forms is similar to that of the vitreous body of the eye. Collagen fibers become a scaffold, and hyaluronic acid is embedded in it to form a tiny gel structure—that is, microgel. The formation of these microgels can not only endow ADM with excellent hydrophilicity, but also "lock" water on the collagen fibers well, and together with the hydrophilicity and moisture retention of collagen itself, a double synergistic effect will be produced, and finally form Microgelled moisturizing type acellular porcine dermal matrix with excellent hydrophilic and moisturizing properties. The formation of microgel structure makes it resistant to water loss and evaporation, thus exhibiting long-lasting hydrophilic and moisturizing properties.

As shown in Figure 6, different amounts of epoxy hyaluronic acid in the present invention still maintain a good microstructure after being cross-linked with ADM. Because epoxy hyaluronic acid is a derivative of hyaluronic acid, the main component is still hyaluronic acid sugar units, thus inheriting the excellent biocompatibility of hyaluronic acid. In addition, the material prepared by the method of the present invention has no skin sensitization and no pyrogen, and the cytotoxicity of its extract is 0-1. Because the epoxy cross-linking reaction adopted in the present invention is less toxic than the traditional aldehyde cross-linking reaction, in addition, even if there is still a small amount of epoxy group remaining in the epoxy cross-linking reaction of the present invention, it will also interact with The amino acid or amino acid salt added later reacts to block the reactivity and eliminate its possible adverse effects on biocompatibility.

The product of the present invention also has higher stability. Because epoxy hyaluronic acid contains multiple epoxy groups, it can undergo cross-linking reactions with amino groups on the acellular dermal matrix. Its cross-linking strength can be adjusted according to needs, thus endowing it with higher stability and better resistance to enzymatic degradation (see Figure 7).

The method of the invention can regulate the preparation process in two aspects of epoxy hyaluronic acid synthesis and cross-linking modification conditions, and prepare materials meeting application requirements. The method of the present invention can also be extended to the preparation of other related materials.

In the present invention, "deionized water" and "deionized water" have the same meaning.

"Pure water" in the present invention includes, for example, primary pure water, secondary pure water, and the like.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually carried out according to conventional conditions.

Main experimental instrument and material of the present invention are as follows:

Table 1 Experimental instruments and equipment

Table 2 Experimental materials and reagents

In some embodiments of the present invention, the amount of EHA, reaction time, reaction temperature, and pH value of the reaction system are respectively optimized by using a single factor method with the surface contact angle as an index. The optimization conditions are as follows:

1. EHA dosage optimization

Control reaction temperature is 37 ℃, and pH value is 10.4, and reaction time is 72h unchanged, and EHA consumption is weighed respectively 0%, 2%, 4%, 8% of pADM dry weight EHA grafted pADM, obtains different dosage rings Oxidized hyaluronic acid-grafted acellular porcine dermal matrix (EHA-pADM).

2. Response time optimization

The reaction temperature was controlled at 37°C, the pH value was 10.4, and the amount of EHA was kept constant at 4%. Different reaction times were selected as 24h, 48h, 72h, and 96h to graft pADM to obtain epoxidized hyaluronic acid grafted with different reaction times. Branched acellular porcine dermal matrix (EHA-pADM).

3. Reaction temperature optimization

The reaction time was controlled to be 72 hours, the pH value was 10.4, and the amount of EHA was kept constant at 4%. Different reaction temperatures were selected as 27°C, 32°C, 37°C, and 42°C to graft pADM to obtain epoxidized transparent Acellular porcine dermal matrix grafted with hyaluronic acid (EHA-pADM).

4. Optimization of the pH value of the reaction system

The reaction temperature is controlled at 37°C, the reaction time is 72h, the amount of EHA is 4%, and the pH values of different reaction systems are selected to be 8.4, 9.4, 10.4, and 11.4 to graft pADM, and the epoxidation with different reaction system pH values is obtained. Hyaluronic acid grafted acellular porcine dermal matrix (EHA-pADM).

5. Contact angle detection

The EHA-grafted pADM material (EHA-pADM) was prepared into a square strip sample of 1.5 cm×7 cm, and the contact angle of the pADM material surface was measured by an optical contact angle measuring instrument. Using the sitting drop method, 6 μl of distilled water was dropped on the surface of the sample, and samples were taken in parallel for 5 times. The contact angle of the sample was calculated on the computer by analyzing the image formed at the moment when the droplet contacted the surface of the pADM sample.

After optimization, the best reaction conditions to obtain EHA-grafted pADM are: the amount of EHA is 8%, the reaction time is 72h, the reaction temperature is 37℃, the pH value of the reaction system is 10.4, and the contact angle of the material obtained under these conditions is 69.82°.

Example 1

(1) Prepare mixed solvent: 1000 grams of distilled water is mixed with 1000 grams of dimethyl sulfoxide to obtain 2000 grams of mixed solvent;

(2) Dissolving hyaluronic acid: dissolve 30 grams of hyaluronic acid in 2000 grams of mixed solvent to obtain a hyaluronic acid solution;

(3) Add catalyst: add 10 grams of sodium borohydride to the hyaluronic acid solution while stirring;

(4) Add epichlorohydrin dropwise: raise the temperature to 60° C., slowly add 5 grams of epichlorohydrin dropwise, and keep warm for 2 hours after the dropwise addition;

(5) Prepare sodium hydroxide solution: take 5 grams of sodium hydroxide, dissolve in 200 grams of water, and cool to normal temperature;

(6) Ring-closing reaction: reduce the temperature of the reaction system to 25°C, add sodium hydroxide solution, and react for 2 hours;

(7) Purification: add 1000 grams of ethanol into the solution after the reaction, let stand for 15 hours, filter the precipitate, dissolve the precipitate with deionized water and pack it into a dialysis bag with a molecular weight cut-off of 8000Da, and immerse in distilled water; Dialyze for 60 minutes, discard the distilled water; then dialyze with distilled water for 4 hours, discard the distilled water; then dialyze in distilled water for 72 hours, during which the distilled water is changed every 24 hours;

(8) Drying: The dialyzed product is taken out, and dried by freeze drying or vacuum drying to obtain epoxy hyaluronic acid.

(9) take by weighing the decellularized porcine dermal matrix of 100g, join in the rotating drum;

(10) adding 200 g of pH is 9.4 sodium carbonate-sodium hydroxide buffer solution, and rotated for 30 minutes;

(11) Take by weighing epoxy hyaluronic acid 5g, join in the rotating drum;

(12) be heated up to 37 ℃, react for 24 hours, pour off waste liquid;

(13) Add 100g of sodium carbonate-sodium hydroxide buffer solution with a pH of 9.4, add 2kg of sodium glutamate (or sodium glutamate), rotate for 60 minutes, and pour off the waste liquid;

(14) Add 200g of deionized water, wash for 30 minutes, and pour off the waste liquid;

(15) Add 100g deionized water, then add 5g ammonium chloride, rotate for 30 minutes, and pour off the waste liquid;

(16) Add 300g of deionized water, wash for 30 minutes, and pour off the waste liquid;

(17) Add 500 g of water for injection and wash for 60 minutes; pour off the cleaning solution, then add 300 g of water for injection and wash for 60 minutes.

(18) Obtaining epoxy hyaluronic acid modified acellular dermal matrix.

Example 2

(1) Prepare mixed solvent: in the head tank, 50Kg distilled water is mixed with 50Kg of 1-4 dioxane to obtain 100kg mixed solvent;

(2) Dissolving hyaluronic acid: add 5Kg hyaluronic acid into the reaction kettle, then add 100Kg mixed solvent, and start stirring for 100 minutes;

(3) Add catalyst: 0.5Kg tetrabutylammonium bromide is added in the hyaluronic acid solution;

(4) Add epichlorohydrin dropwise: raise the temperature of the hyaluronic acid solution to 70°C, slowly add 2Kg of epichlorohydrin dropwise, and keep warm for 2 hours after the dropwise addition;

(5) Potassium hydroxide solution preparation: take by weighing the potassium hydroxide of 0.5Kg, be dissolved in the dehydrated alcohol of 10Kg, be cooled to normal temperature;

(6) Ring-closing reaction: reduce the temperature of the reaction system to 15° C., add potassium hydroxide solution, and react for 2 hours;

(7) Purification: add 100Kg ethanol into the solution after the reaction, let stand at low temperature for 24 hours, filter the precipitate, dissolve the precipitate with deionized water and pack it into a dialysis bag with a molecular weight cut-off of 500Da, and immerse in distilled water; Dialyze for 60 minutes, discard the distilled water; then dialyze with distilled water for 240 minutes, discard the distilled water; then dialyze in distilled water for 72 hours, during which the distilled water is changed every 24 hours;

(8) Drying: take out the reactant after dialysis, and adopt freeze-drying method or vacuum drying method to dry it to obtain epoxy hyaluronic acid.

(9) take by weighing the decellularized bovine dermis matrix of 1kg, join in the drum;

(10) adding 3 kg of pH is 10.4 sodium carbonate-sodium hydroxide buffer solution, and rotates for 30 minutes;

(11) Weigh 0.2kg of epoxy hyaluronic acid and add it to the drum;

(12) be warming up to 30 ℃, react for 48 hours, pour off waste liquid;

(13) Add 3kg of ion-free water, wash for 20 minutes, and pour off the waste liquid;

(14) Add 3kg of sodium carbonate-sodium hydroxide buffer solution with a pH of 10.4, add 0.05kg of sodium glutamate, rotate for 60 minutes, and pour off the waste liquid;

(15) Add 3kg of deionized water, wash for 30 minutes, and pour off the waste liquid;

(16) Add 2kg of deionized water, then add 30g of ammonium chloride, rotate for 30 minutes, and pour off the waste liquid;

(17) Add 6kg of deionized water, wash for 30 minutes, and pour off the waste liquid;

(16) Add 6 kg of water for injection at 37°C and wash for 60 minutes; pour off the cleaning solution, then add 6 kg of water for injection and wash for 60 minutes.

(18) Obtaining epoxy hyaluronic acid modified acellular dermal matrix.

Example 3

(1) Preparation of mixed solvent: mix 50Kg of distilled water with 50Kg of N,N-dimethylformamide to obtain 10Kg of mixed solvent;

(2) Dissolving hyaluronic acid: 2kg of hyaluronic acid is dissolved in a mixed solvent of 100Kg to obtain a hyaluronic acid solution;

(3) Add catalyst: 100g sodium borohydride is added in the hyaluronic acid solution;

(4) Add epoxy organic matter dropwise: raise the temperature of the hyaluronic acid solution to 65°C, slowly add 0.7Kg of epichlorohydrin dropwise, and keep warm for 3 hours after the dropwise addition;

(5) prepare sodium hydroxide solution: take by weighing the sodium hydroxide of 0.4kg, be dissolved in the water of 20Kg, be cooled to normal temperature;

(6) Ring-closing reaction: reduce the temperature of the reaction system to 20°C, add sodium hydroxide solution, and react for 2 hours;

(7) Purification: add 100Kg ethanol into the solution after the reaction, let stand at low temperature for 20 hours, filter the precipitate, dissolve the precipitate with deionized water and pack it into a dialysis bag with a molecular weight cut-off of 3000Da, and immerse in distilled water; Dialyze for 60 minutes, discard the distilled water; then dialyze with distilled water for 240 minutes, discard the distilled water; then dialyze in distilled water for 72 hours, during which the distilled water is changed every 24 hours;

(8) Drying: take out the reactant after dialysis, and adopt freeze-drying method or vacuum drying method to dry it to obtain epoxy hyaluronic acid.

(9) take by weighing the decellularized fetal bovine dermal matrix of 10kg, join in the rotating drum;

(10) adding 20 kg of pH is 10.4 sodium carbonate-sodium hydroxide buffer solution, and rotates for 30 minutes;

(11) Weigh 0.5kg of epoxy hyaluronic acid and add it to the drum;

(12) be heated up to 42 ℃, react for 24 hours, pour off waste liquid;

(13) Add 10kg of sodium carbonate-sodium hydroxide buffer solution with a pH of 10.4, add 0.2kg of sodium aspartate (or sodium aspartate), rotate for 60 minutes, and pour off the waste liquid;

(14) Add 20kg of deionized water, wash for 30 minutes, and pour off the waste liquid;

(15) Add 10kg of deionized water, then add 0.25kg of ammonium chloride, rotate for 30 minutes, and pour off the waste liquid;

(16) Add 30kg of deionized water, wash for 30 minutes, and pour off the waste liquid;

(17) Add 30kg of water for injection and wash for 60 minutes; pour off the cleaning solution, then add 40kg of water for injection and wash for 60 minutes.

(18) Obtaining epoxy hyaluronic acid modified acellular dermal matrix.

Embodiment 4 results and discussion

As shown in Fig. 3, the properties of the epoxidized hyaluronic acid (EHA) obtained by the method of the present invention are white sponge and the properties of hyaluronic acid (HA) are white powder. It can be seen from Fig. 8 that the absorption peaks at 1151cm ^-1 , 1078cm ^-1 and 1047cm ^-1 are characteristic absorption peaks of sugar rings in HA and EHA. The absorption peak at around 940cm ^-1 is the characteristic absorption peak of the epoxy group, and the peak shape here becomes stronger after the reaction, indicating that a new epoxy bond is formed. It can be seen from Figure 9 that, compared with HA NMR, EHA has three new peaks at δ4.36, δ3.49, and δ3.40EHA, corresponding to a, b, and c hydrogens in the formula. The epoxy value of the EHA prepared by the method of the present invention can reach 0.20-0.25mol/100g as measured by the sodium thiosulfate method.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the appended claims of the present application.

Claims (16)

1. a preparation method of epoxy hyaluronic acid modified acellular dermal matrix, is characterized in that, comprises the following steps: (1) Preparation of epoxy hyaluronic acid: dissolve hyaluronic acid in a mixed solvent, add a catalyst, add epichlorohydrin to carry out a ring-opening reaction; then in an alkali solution, carry out a ring-closing reaction; then purify and dry to obtain epoxy hyaluronic acid; (2) Modified cross-linking: the acellular dermal matrix material is modified and cross-linked with the epoxy hyaluronic acid prepared in step (1) to obtain an epoxy hyaluronic acid modified acellular dermal matrix. 2. the preparation method according to right 1, is characterized in that, in step (1), described mixed solvent is the mixed solvent of organic solvent and water, and described organic solvent is selected from dimethylsulfoxide, 1,4- One or more of dioxane, N,N dimethylformamide, ether or acetonitrile, the volume ratio of the organic solvent to water in the mixed solvent is 1:1-1:1.5. 3. preparation method according to right 1, is characterized in that, in step (1), described catalyst is sodium borohydride or tetrabutylammonium bromide. 4. The preparation method according to claim 1, characterized in that, in step (1), the temperature of the ring-opening reaction is 30-80°C, and the time of the ring-opening reaction is 2-8 hours. 5. The preparation method according to claim 1, characterized in that, in step (1), the alkali solution is 2-50% aqueous or ethanol solution of sodium hydroxide or potassium hydroxide. 6. The preparation method according to claim 1, characterized in that, in step (1), the temperature of the ring-closing reaction is 4-25° C., and the reaction time is 1-4 hours. 7. The preparation method according to claim 1, characterized in that in step (1), the purification comprises the following steps: adding ethanol to the reacted solution, and standing at 0-5°C for 12-48 hours Collect the precipitate, dissolve the precipitate with deionized water, put it into a dialysis bag, immerse in distilled water for dialysis, and collect the dialysis product. 8. The preparation method according to claim 7, characterized in that, the molecular weight cut-off of the dialysis bag is 500-14000 Da, and the dialysis method is: first dialysis for 60-120 minutes, discarding the distilled water; then adding distilled water for dialysis for 240 ~360 minutes, discard the distilled water; then dialyze in distilled water for 72 hours, during which the distilled water is changed every 24 hours. 9. The preparation method according to claim 1, characterized in that, in step (1), the drying adopts a freeze-drying method or a vacuum drying method. 10. The preparation method according to claim 1, characterized in that, in step (1), based on 100 parts by weight of hyaluronic acid, the amount of the mixed solvent added is 500 to 5000 parts by weight, and the amount of the catalyst is The added amount is 5-50 parts by weight, the added amount of the epichlorohydrin is 20-100 parts by weight, the alkali solution is 50-500 parts by weight, and the added amount of ethanol is 500-2000 parts by weight. 11. The preparation method according to claim 1, characterized in that in step (2), the modified cross-linking comprises the following steps: weighing 100 parts by weight of the acellular dermal matrix material, adding 100 ～1000 parts by weight of buffer solution with a pH of 7.0～10.5, soaked for 10～60min; then add 1～50 parts by weight of epoxy hyaluronic acid obtained in step (1), and process for 12～72 hours; discard the waste liquid, add Wash with 200-1000 parts by weight of water for 10-30 minutes; discard the cleaning waste liquid, add 100-1000 parts by weight of a buffer solution with a pH of 7.0-10.5, add 1-10 parts by weight of amino acids or amino acid salts, and heat at 25-47 ° C Down treatment of 30-120mim; discard the waste liquid, add 200-1000 parts by weight of pure water to wash for 10-30min; discard the waste liquid, add 200-1000 parts by weight of pure water to wash for 10-30min; discard the waste liquid, add 5-10 parts by weight of ammonium chloride or lactic acid, react for 30-60 minutes; discard the waste liquid, add 200-1000 parts by weight of water for injection at 20-40°C, and wash for 60-120 minutes; discard the waste liquid, add 200-1000 Injection water at a temperature of 20-40° C. is used in parts by weight, and washed for 60-120 minutes; an epoxy hyaluronic acid modified acellular dermal matrix is obtained. 12. The preparation method according to claim 11, wherein the acellular dermal matrix is selected from one of acellular porcine dermal matrix, acellular fetal bovine dermal matrix, acellular goat dermal matrix and acellular bovine dermal matrix. one or more; the buffer solution is a phosphate buffer solution, a sodium carbonate-sodium bicarbonate buffer solution or a sodium carbonate-sodium hydroxide buffer solution; the amino acid is glutamic acid or aspartic acid, and the amino acid salt For sodium glutamate or sodium aspartate. 13. The epoxy-hyaluronic acid-modified acellular dermal matrix prepared by the preparation method according to claims 11-12. 14. The epoxy-hyaluronic acid-modified acellular dermal matrix according to claim 13, characterized in that the shrinkage temperature of the epoxy-hyaluronic acid-modified acellular dermal matrix is 62-78°C, and the contact angle is 10-50°, the moisture absorption rate is 400-600%, the water retention rate is 150-300%, the water loss rate is 20-40%, the equilibrium adsorption capacity is 250-400mg/g, and the cytotoxicity of the extract is 0-1 . 15. The use of the epoxy hyaluronic acid modified acellular dermal matrix according to claims 13-14 in the preparation of moisturizing dressings. 16. The epoxy hyaluronic acid prepared in the step (1) of the preparation method according to claims 11-12.