WO2014101748A1 - 胶原止血或抗凝材料 - Google Patents

胶原止血或抗凝材料 Download PDF

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WO2014101748A1
WO2014101748A1 PCT/CN2013/090324 CN2013090324W WO2014101748A1 WO 2014101748 A1 WO2014101748 A1 WO 2014101748A1 CN 2013090324 W CN2013090324 W CN 2013090324W WO 2014101748 A1 WO2014101748 A1 WO 2014101748A1
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collagen
amino acid
reaction
group
solution
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PCT/CN2013/090324
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French (fr)
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杨军
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上海欣吉特生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides

Definitions

  • the invention relates to a collagen material and a preparation method thereof, in particular to a collagen material having local hemostasis or anticoagulant function and a preparation method thereof.
  • Collagen is the most abundant protein in many vertebrate and invertebrate organisms. It is a structural protein. It is the main fiber component of skin, bone, cartilage, blood vessels and teeth. It is found in all organs. There are currently 28 identified collagens, including from type I to XXVIII. Among them, type I collagen is the most abundant in animal body and its immunogenicity is much lower than other types of collagen, so it is also widely used.
  • the basic structure of collagen is composed of three The alpha polypeptide chain is composed of each ⁇ chain itself in a left-handed helical configuration, and the three ⁇ chains are entangled with each other to form a right-handed supercoiled structure.
  • Each alpha chain is made up of multiple alpha-
  • the amino acid dehydration condensation forms a peptide bond (-NH-CO-), and the usual peptide segment amino acids are periodically arranged (Gly-X-Y), and each peptide chain has about 1000 amino acid residues.
  • ⁇ -amino acids there are more than 30 kinds of amino acids in nature, among which There are 20 kinds of ⁇ -amino acids, only ⁇ -amino acids can constitute natural proteins, while natural collagen contains most of the ⁇ -amino acids.
  • the structural difference of the ⁇ -amino acid depends on the side chain group R, according to the R group Polarity or chemical structure can classify ⁇ -amino acids as: 1.
  • Non-polar amino acids also called hydrophobic amino acids 8 species: alanine (Ala), proline (Val), leucine (Leu), Isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met); 2, polar amino acids (also known as hydrophilic amino acids) It is further divided into: 1) 7 kinds of polar uncharged amino acids: Glycine (Gly), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln); 2 3 kinds of polar positively charged amino acids (also called basic amino acids): Lysine (Lys), arginine (Arg), histidine (His); 3) Polar negatively charged amino acids ( Can also be called Acidic amino acids) 2 kinds: aspartic acid (Asp), glutamic acid (Glu).
  • polar uncharged amino acids Glycine (Gly),
  • the lysine content is about 3%
  • the total aspartic acid and glutamic acid content is about 13%
  • the arginine and histidine content are small
  • the polar amino acid residues are relatively active and easy. Participate in various chemical reactions.
  • the composition and arrangement sequence of amino acids determine the primary structure of the peptide chain, which leads to differences in macromolecular morphology, which in turn leads to differences in collagen structure and ultimately results in differences in biological properties.
  • hemostatic materials commonly used in clinical practice such as hemostatic gauze, hemostatic fiber, and hemostatic bandage have limitations in use: hemostasis time is long, easy to adhere to the wound and not easy to change the medicine, and it is powerless to the infection and suppuration of the wound. Therefore, people continue to develop new hemostatic materials, mainly including: gelatin sponge, chitosan, oxidized cellulose, fibrin glue, calcium alginate fiber, microporous inorganic materials (such as zeolite), microfiber collagen and so on. The mechanism of action and use of these new hemostatic materials are not the same, so the hemostatic effect is also different. Proper hemostatic material can significantly shorten the operation time and is essential for trauma or postoperative recovery.
  • Fibrin glue can form fibrin clots to adhere to the wound surface, which can reduce the chance of wound hematoma formation. It is suitable for hemorrhage of retroperitoneal hemorrhage and abdominal organ hemorrhage.
  • Oxidized regenerated cellulose has the effect of inhibiting bacteria and preventing postoperative adhesion, and is suitable for hemostasis of intra-abdominal organs, but should be avoided in neurosurgery surgery, and the operation is complicated and the cost is high.
  • Gelatin sponges rapidly expand in volume after absorbing blood, compressing surrounding tissues, and should be avoided in areas close to nerves or small spaces.
  • Microfibrous collagen hemostasis requires platelet involvement and is not suitable for patients with platelet deficiency.
  • the normal hemostasis mechanism consists of three main processes: contraction of small blood vessels, activation of platelets, and initiation of the coagulation system. Coagulation is divided into two pathways: endogenous coagulation and exogenous coagulation, but usually the two pathways are not clearly differentiated during the actual coagulation process.
  • various components in the blood such as plasma proteins, hemoglobin on red blood cells, etc.
  • aggregation may occur when exposed to positively charged foreign matter, thereby promoting coagulation.
  • the hemostatic mechanism of biomaterials made of collagen is mainly through the action of platelets to achieve hemostasis.
  • the specific realization method is as follows: when the material is in contact with blood, the surface of the material rapidly forms a protein adsorption layer, and then causes The aggregation of platelets on the surface of the material causes the platelets to release some subcellular particles and secretions, including various coagulation factors. The coagulation factors adhere to the wound, causing thrombosis, filling the injured blood vessels, and achieving hemostasis.
  • the ideal hemostatic material should have the following characteristics: rapid hemostasis, non-toxicity, no antigenicity, no increase in infection probability, no effect on tissue healing, not easy to dissolve, but rapid degradation and adjustable, low price.
  • collagen has unparalleled superiority in synthetic biomaterials, health care materials, etc., and the undenatured collagen obtained by various methods has its structure still retaining its natural structure in animals. Studies have shown that collagen has the advantages of low immunogenicity, biodegradability, hemostasis, etc.
  • collagen-based hemostatic materials such as collagen sponge, gelatin, etc. usually make natural collagen into a gel solution or collagen extract, which often destroys the spiral structure and biological activity of natural collagen during its preparation.
  • Simple gelatin sponge or collagen sponge has poor toughness, is not tightly combined with wound surface, and has no anti-edema effect.
  • tissue engineering scaffold material Because of its good biocompatibility, it is often used as a tissue engineering scaffold material. It has been widely used to repair damaged tissues in human body. However, some parts such as blood vessels, heart valves, and atrioventricular septum are damaged.
  • the repair material is required to have appropriate anticoagulant properties to prevent thrombosis.
  • vascular repair materials, biological heart valves made from bovine pericardium need to have good blood compatibility. Since thrombus formation mainly occurs on the surface of the material, as long as any part of the thrombus is inhibited or blocked, good anticoagulant properties can be obtained, so the surface modification of the material can be considered from the viewpoint of mutual regulation of the surface of the material and blood.
  • the commonly used methods are: 1.
  • the surface of the material is negatively charged, because the blood components are in the blood environment. Negatively charged, using electrostatic repulsion to block the adsorption of plasma proteins and other substances; 3, the surface of the material is biologicalized, the bioactive substance with anticoagulant function is loaded on the surface of the material; 4, the surface of the material is endothelialized, and the endothelial cells are cultured on the surface of the material. It eventually grows a layer of endothelium.
  • the present invention seeks to retain the original helical region structure of natural collagen, and to change the electrical properties by reacting specific groups on the collagen fibers, thereby designing a collagen material having a hemostatic function or an anticoagulant function. Meet different needs.
  • the object of the present invention is to provide a novel collagen material having local hemostasis or anticoagulant function, which is designed by reacting a specific amino acid polar group such as a carboxyl group or an amino group on a collagen peptide chain to change its electrical properties.
  • the collagen material provided by the invention can be implanted to locally stop bleeding or anticoagulant, and can be used for cells around the damaged part to grow therein to promote healing of the damaged tissue.
  • the present invention adopts the following technical solutions:
  • a method of preparing a collagen material having local hemostasis or anticoagulant function comprising the steps of:
  • the positively charged amino acid group is introduced into the carboxyl group of the acidic amino acid on the collagen peptide chain by the participation of the epoxy compound and the basic catalyst to positively charge the material;
  • the basic catalyst here may be pyridine, isoquine Compounds such as porphyrins have a concentration ranging from 0.1% to 10% (v/v).
  • the activator By activating the carboxyl group on the polar negatively charged amino acid in the collagen, causing it to undergo a condensation reaction with the free amino group added to the solution, thereby reducing the negative charge on the collagen substrate, thereby positively charging the material; the activator here can It is a carbon diamine compound having a concentration ranging from 0.1% to 5% (w/v), and the added free amino group may be a polar amino acid.
  • the surface of the material is chemically treated for a short time to make the surface electrical properties and internal electrical properties different.
  • the chemical treatment here mainly destroys the ester bond formed by the reaction in 1-5, and hydrolyzes it to -COO - in a time range of 5-30 minutes.
  • the chemical reaction in the step 1 is an aldehyde or an epoxy compound and ⁇ on the lysine in the collagen.
  • the aldehyde is glutaraldehyde, more preferably the glutaraldehyde has a volume concentration of from 0.1% to 2%; preferably the epoxy compound is ethylene glycol diglycidyl ether and/or polypropylene oxide.
  • the step 1 is specifically: Collagen and glutaraldehyde (volume concentration 0.1%-2%) were reacted at pH 6.0-8.0.
  • the reaction process was as shown in Figure 2, and the reaction time ranged from 30 minutes to 14 days.
  • the prepared collagen substrate was prepared. Negatively charged in solution, with anticoagulant properties; for example, placing bovine pericardium with added pH 7.4
  • the PBS buffer was dissolved in 0.6% (v/v) glutaraldehyde solution for 3 days.
  • alkaline conditions pH 8.0-10.5
  • the collagen is reacted with an epoxy compound, and the epoxy group and the lysine in the epoxy compound are ⁇ - The amino group reacts.
  • the reaction process is shown in Figure 3.
  • the reaction time ranges from 1 hour to 14 days.
  • the collagen substrate is negatively charged in the solution and has anticoagulant properties; for example, the bovine pericardium is placed in the addition.
  • the chemical reaction in step 2 is the reaction of ethylene glycol diglycidyl ether with a carboxyl group on a polar negatively charged amino acid in collagen under the action of a basic catalyst; collagen and ethylene glycol diglycidyl ether
  • the dosage is 1 g of collagen added to 50 mL, and the basic catalyst is pyridine or isoquinoline at a concentration ranging from 0.1% to 10% (v/v).
  • the step 2 is specifically: reacting collagen such as bovine pericardium with 2% (v/v) ethylene glycol diglycidyl ether in a ratio of 1 g: 50 mL under the catalysis of 0.5% (v/v) pyridine. .
  • the reaction time is from 1 hour to 14 days. After the reaction is completed, the collagen substrate is positively charged in the solution and has a property of promoting coagulation.
  • the epoxy compound A in the step 3 is ethylene glycol diglycidyl ether and/or polypropylene oxide, and the polar charged amino acid group is lysine, aspartic acid and/or valley. Amino acid.
  • the step 3 is specifically: collagen and glutaraldehyde (volume concentration 0.1%-2%) and lysine (mass concentration) under weakly acidic to weakly alkaline conditions (Ph6.0-8.0) 1%) a volume ratio of 1:1 mixed solution reacts here,
  • the ratio of collagen to mixed solution is: 1g collagen plus mixed solution 50mL,
  • the reaction process is shown in Figure 4.
  • the reaction time ranges from 30 minutes to 14 days; or under a basic condition of pH 8.0 to 10.5, a reaction mixture of collagen and ethylene glycol diglycidyl ether: lysine is 1:1, B Two epoxy groups in diol diglycidyl ether and ⁇ of lysine in collagen peptide chain - Amino and ⁇ of lysine in solution - The amino group reacts and the lysine in the solution is introduced into the collagen substrate for a reaction time ranging from 1 hour to 14 days.
  • the reaction process is shown in Figure 4. After the reaction is completed, the collagen substrate is negatively charged in the solution and has anticoagulant properties.
  • the polar positively charged amino acid group in step 4 is lysine
  • the epoxy compound is ethylene glycol diglycidyl ether and/or polypropylene oxide.
  • the ratio of collagen such as bovine pericardium to lysine and ethylene glycol diglycidyl ether is 1:2 (w/w) under the catalysis of 0.5% (v/v) pyridine.
  • the reaction was carried out in a mixed solution, and the reaction process is shown in FIG. The reaction time is from 1 hour to 14 days. After the reaction is completed, the collagen substrate is positively charged in the solution and has a property of promoting coagulation.
  • the polar amino acid in step 5 is lysine.
  • EDC carboxysuccinimide
  • EDC carboxysuccinimide
  • NHS N- a mixture of hydroxysuccinimide (EDC: NHS mass ratio of 5:1
  • the COOH reacts to form an unstable O-isoacylurea structure
  • the NHS forms a stable ester group and reacts with the free amino group in the solution.
  • the reaction process is shown in Figure 5. After the reaction is completed, the entire collagen substrate is positively charged in
  • placing calfskin in contains 1% (w/w) lysine, pH 5.5 - 6.7 MES buffer, carbodiimide: a solution having a NHS ratio of 5:1 (w/w) for 1-3 hours, and a carbodiimide concentration of 1% (w/v).
  • step 6 is: immersing collagen, such as bovine pericardium, in a ratio of lysine to ethylene glycol diglycidyl ether of 1:2 (w/w) under the catalysis of 0.5% (v/v) pyridine.
  • the reaction is carried out in the mixed solution for 14 days, and then placed under weakly acidic conditions (pH 5.5-6.5) for 5-10 minutes to hydrolyze the ester bond on the collagen substrate close to the inserted lysine group to make the collagen group.
  • the surface of the material is restored to neutral or slightly negative, while the interior of the substrate is still positively charged.
  • the collagen of the present invention is: animal tissue collagen, such as bovine pericardium, calfskin.
  • the beneficial effects of the invention are as follows: (1) not destroying the structure of the natural collagen helix region, retaining its biological activity, and is more favorable for tissue adsorption and growth than the existing collagen hemostatic sponge; (2) by changing the collagen substrate electricity It can promote coagulation or anticoagulant to meet different needs.
  • a is a Lys structure
  • b is an Asp structure
  • c is a Glu structure
  • Figure 2 is a process in which a carboxyl group on aspartic acid in a collagen reacts with an epoxy compound under the action of a basic catalyst;
  • Figure 3 shows the reaction of ⁇ -amino group on lysine in collagen with glutaraldehyde
  • Figure 4 shows the reaction of ⁇ -amino group on lysine in collagen with epoxy compound under alkaline conditions
  • Figure 5 shows the reaction between glutaraldehyde and the ⁇ -amino group on lysine in collagen and the amino group on lysine in solution under weakly acidic to weakly alkaline conditions.
  • Figure 6 shows the reaction between epoxy compound and ⁇ -amino group on lysine in collagen and amino group on lysine in solution under alkaline conditions.
  • Figure 7 shows the reaction of the carboxyl group on aspartic acid in the collagen with the epoxy compound and the added lysine under the action of a basic catalyst
  • Figure 8 shows the reaction of the carboxyl group on aspartic acid in collagen with the carbodiimide/NHS solution.
  • the bovine pericardium is placed in a solution of sodium carbonate-sodium bicarbonate buffer of pH 8.0-10.5 in ethylene glycol diglycidyl ether for 9 days, where the sodium carbonate-sodium bicarbonate buffer is 0.04 mol. /L, ethylene glycol diglycidyl ether content 0.02% (v / v).
  • the materials obtained by the above two methods were each taken in 1 mL 2 and placed in 5 mL of plasma supplemented with sodium citrate. A few drops of 1 mol/L CaCl 2 solution were added, and the coagulation reaction began to appear in 8-9 minutes. 5-6 minutes.
  • the bovine pericardium was reacted with 2% (v/v) ethylene glycol diglycidyl ether in a ratio of 1 g: 50 mL under the catalysis of 0.5% (v/v) pyridine.
  • the reaction time is from 1 hour to 14 days.
  • the collagen substrate is positively charged in the solution and has the property of promoting coagulation.
  • the obtained material is taken in 1 mL 2 and placed in 5 mL of plasma with sodium citrate, and then a few drops of 1 mol/L CaCl 2 solution, 3
  • the coagulation reaction begins in 4 minutes, and normal coagulation takes 5-6 minutes.
  • the mixed solution reacts, where the ratio of collagen to mixed solution is: 1 g of collagen plus mixed solution 50 mL, and the reaction process is shown in Fig. 4.
  • the reaction time ranges from 30 minutes to 14 days; or ethylene glycol diglycidyl ether in sodium carbonate-sodium bicarbonate buffer under alkaline conditions such as pH 8.0-10.5, here, sodium carbonate-sodium bicarbonate buffer
  • the solution is 0.04 mol/L, and the ethylene glycol diglycidyl ether content is 0.02% (v/v).
  • the mixture of collagen and ethylene glycol diglycidyl ether: lysine is 1:1 (w/w).
  • two epoxy groups in ethylene glycol diglycidyl ether react with the ⁇ -amino group of lysine in the collagen peptide chain and the ⁇ -amino group of lysine in the solution, respectively, and the lysine in the solution is connected.
  • the reaction time ranges from 1 hour to 14 days. The reaction process is shown in Figure 5.
  • the collagen substrate is negatively charged in the solution, and the obtained material is taken in 1 mL 2 in 5 mL of plasma supplemented with sodium citrate, and then a few drops of 1 mol/L CaCl 2 solution are added, and 8-10 minutes later. Coagulation occurs and normal coagulation takes 5-6 minutes.
  • the bovine pericardium is immersed in a mixed solution of lysine and ethylene glycol diglycidyl ether in a ratio of 1:2 (w/w), and the reaction process is as follows.
  • Figure 6 shows.
  • the reaction time is from 1 hour to 14 days.
  • the collagen substrate is positively charged in the solution and has the property of promoting coagulation.
  • the obtained material is taken in 1 mL 2 and placed in 5 mL of plasma with sodium citrate, and then a few drops of 1 mol/L CaCl 2 solution, 3
  • the coagulation reaction begins in 4 minutes, and normal coagulation takes 5-6 minutes.
  • the bovine pericardium was immersed in a mixed solution of lysine and ethylene glycol diglycidyl ether in a ratio of 1:2 (w/w) for 14 days, then The MES buffer at pH 5.5-6.5 is placed for 5-10 minutes to hydrolyze the ester bond on the collagen substrate near the lysine group, so that the surface of the collagen substrate is restored to neutral or slightly negative. The inside of the substrate is still positively charged. The resulting material is taken 1cm 2 was placed sodium citrate added 5mL plasma, and then a few drops of 1mol / L of CaCl 2 solution, the reaction product after coagulation for pathological sections were observed inside the collagen have focused on blood clots.

Abstract

本发明提供了一种具有局部止血或抗凝血功能的胶原材料，通过对胶原肽链上的氨基酸极性基团羧基或氨基进行反应，改变其电性，从而得到具有促进凝血的局部止血材料或防止凝血的抗凝血材料；本发明所提供的胶原材料可进行植入，起到局部止血或抗凝血作用，并可供受损部位周围的细胞进入其中生长，促进受损组织愈合。

Description

胶原止血或抗凝材料
技术领域
本发明涉及一种胶原 材料及其制备方法，特别涉及一种具有局部止血或抗凝血功能的胶原材料及其制备方法。
背景技术
胶原是许多脊椎动物和无脊椎物动体内含量最为丰富的蛋白质，属于结构蛋白质，是皮肤，骨，软骨、血管和牙齿的主要纤维成分，存在于所有的器官中。目前已确认的胶原有28种，包括从Ⅰ型到ⅩⅩVIII型。其中Ⅰ型胶原在动物体内含量最多并且其免疫原性比其他型胶原要低得多，因而被运用的也最为广泛。胶原基本结构为由三条 α 多肽链组成，每一条 α 链自身为左手螺旋构型，三条 α 链相互缠绕在一起形成右手超螺旋结构。每一条 α 链是由多个 α- 氨基酸脱水缩合形成肽键（-NH-CO-）连接而成，通常的肽链段氨基酸呈（Gly-X-Y）周期性排列，每条肽链约有1000个氨基酸残基。已知自然界中共有30多种氨基酸，其中 α- 氨基酸有20种，只有 α- 氨基酸才能构成天然蛋白质，而天然胶原中基本都含有大部分 α- 氨基酸。 α- 氨基酸结构上差别取决于侧链基团R，根据R基团的 极性 或化学结构可将 α- 氨基酸分类为： 1 、非极性氨基酸（也可称疏水氨基酸）8种： 丙氨酸（Ala） 、 缬氨酸（Val） 、 亮氨酸（Leu） 、 异亮氨酸（Ile） 、 脯氨酸（Pro） 、 苯丙氨酸（Phe） 、 色氨酸（Trp） 、 蛋氨酸（Met） ； 2 、极性氨基酸（ 也可称 亲水氨基酸） 又分为 ：1）极性不带电荷 的氨基酸 7 种 ： 甘氨酸（Gly）、丝氨酸（Ser）、苏氨酸（Thr）、半胱氨酸（Cys）、酪氨酸（Tyr）、天冬酰胺（Asn）、谷氨酰胺（Gln） ； 2 ）极性带正电荷的氨基酸（ 也可称 碱性氨基酸）3种 ： 赖氨酸（Lys）、精氨酸（Arg）、组氨酸（His） ； 3 ）极性带负电荷的氨基酸（ 也可称 酸性氨基酸） 2种 ： 天冬氨酸（Asp）、谷氨酸（Glu） 。胶原中所含有的 极性氨基酸中，赖氨酸含量约为3%，天冬氨酸和谷氨酸总含量约为13%，精氨酸和组氨酸含量很少，且极性氨基酸残基较为活泼，容易参加各种化学反应。氨基酸的组成和排布序列决定了肽链的初级结构，从而导致大分子形态差异，进而造成各型胶原结构差异，最终造成生物学性能差异。
在伤口急救以及外科手术中，创面出血与渗血是经常遇到的难题，血液的大量流失会危及生命，同时血液是天然的微生物培养基，出血或渗血都会增加感染几率，从而降低手术成功率，因此，外科止血是手术技术核心之一。单纯依靠机械性止血方法如结扎、缝合、电烙等还不够，还需配合使用止血材料的来帮助止血。临床常用的传统止血材料如止血纱布、止血纤维、止血绷带在使用中都具有局限性：止血时间较长，易与伤口粘连而不易换药，对伤口的感染和化脓无能为力。因此人们又不断开发出新型的止血材料，主要有：明胶海绵，壳聚糖，氧化纤维素，纤维蛋白胶，藻酸钙纤维，多微孔类无机材料（例如沸石），微纤维胶原等。这些新型止血材料的作用机制和使用方法不尽相同，因此止血效果也有差别。合适的止血材料能明显缩短手术时间，对创伤或术后恢复至关重要。在选择止血材料时应当多方面综合考虑，包括出血部位，术腔形态大小，不同渗/出血情况的填塞要求，不同止血材料的止血机制，与机体的相容性，患者的凝血功能和经济条件等。纤维蛋白胶类能形成纤维蛋白凝块黏附于创面，可减少创面血肿形成的机会，适用于腹膜后出血，腹腔脏器出血的止血。氧化再生纤维素类具有抑菌和防止术后粘连的作用，适用于腹腔内脏器的止血，但在神经外科的手术中应避免使用，且操作复杂，成本较高。明胶海绵在吸收血液后体积迅速膨胀，压迫周围组织，应避免在靠近神经或空间狭小的部位使用。微纤维胶原止血过程需要血小板参与，不适用于血小板缺乏症患者。
正常的止血机理主要包括三个过程：小血管的收缩，血小板的激活和凝血系统的启动。凝血又分为内源性凝血和外源性凝血两条途径，但通常在实际凝血过程中这两条途径区分并不明显。此外，由于血液中的多种组分（如血浆蛋白，红细胞上的血红蛋白等）在血液环境中呈负电性，一旦接触到带正电荷异物易发生聚集，可起到促进凝血作用。目前利用胶原制成的生物材料（例如胶原海绵）的止血机理主要是通过与血小板作用来达到止血目的，具体实现方式为：材料在与血液接触时，其表面迅速形成了蛋白吸附层，而后引起血小板在材料表面上的聚集粘附，刺激血小板释放一些亚细胞颗粒和分泌物，包括各种凝血因子，凝血因子粘附在伤口上，导致血栓形成，填塞受伤血管，达到止血目的。
新型理想的外科手术补片也希望具有局部止血功能。理想的止血材料应具备以下特点：止血迅速、无毒、无抗原性、不增加感染概率、不影响组织愈合、不易溶解但降解速度较快且可调、价格低廉。近年来随着对胶原研究的不断深入，特别是基因工程和克隆技术的飞跃发展，人们对胶原的形态结构、分布、物理性质以及生物生理功能有了更深刻的了解。胶原在医用生化材料、保健材料等方面，具有合成材料不可比拟的优越性，用各种不同方法精制得到的未变性胶原，其结构仍然保持其在动物体内时的天然结构。研究表明，胶原具有低免疫原性，可生物降解性，止血功能等作为天然生物材料的优势，并且体内易被吸收，无毒无刺激。正因胶原表现出良好的止血作用，目前已被大量应用于制备止血海绵。不过目前以胶原为原料的止血材料如胶原海绵、明胶等通常是将天然胶原制成胶溶液或胶原提取物，往往在其制备过程中破坏了天然胶原的螺旋区结构和生物活性。单纯明胶海绵或胶原蛋白海绵的韧性较差，与创面结合不紧，且无抗水肿效果。
而另一方面，胶原因其良好的生物相容性常用作组织工程支架材料，已被广泛用于修复人体受损组织，然而有些部位如血管、心脏瓣膜、心脏房室间隔等受损时，需要修复材料具有适当的抗凝血性以防止血栓发生。例如，血管修复材料，利用牛心包制成的生物心脏瓣膜就需要具有良好的血液相容性。由于血栓形成主要发生在材料表面，只要形成血栓的任何一个环节受到抑制或阻断，都可得到良好的抗凝血性，故可考虑从材料表面与血液相互调控的角度来对材料表面改性。目前常用的方法有：1、对材料表面接枝改性，减少材料与血液成分的相互作用，阻抗血浆蛋白吸附；2、使材料表面带有负电荷，因血液多种组分在血液环境中呈负电性，利用静电排斥作用阻碍血浆蛋白等物质吸附；3、材料表面生物化，将具有抗凝血功能的生物活性物质负载到材料表面；4、材料表面内皮化，在材料表面培养内皮细胞使其最终生长出一层内皮。
大量对生物材料抗凝血作用的研究表明，表面带负电荷的材料具有较好的抗凝血作用。此外，有研究表明未破坏天然螺旋区结构的胶原比已破坏的胶原对细胞有吸附和促进细胞生长作用。因此，本发明寻求保留天然胶原原有的螺旋区结构的基础上，通过对胶原纤维上特定的基团进行反应，改变其电性，从而设计出具有止血功能或抗凝血功能的胶原材料以满足不同需求。
发明内容
本发明的目的在于提供一种新的具有局部止血或抗凝血功能的胶原材料，通过对胶原肽链上特定的氨基酸极性基团如羧基或氨基进行反应，改变其电性，从而设计出具有促进凝血的局部止血材料或防止凝血的抗凝血材料。本发明所提供的胶原材料可进行植入，起到局部止血或抗凝血作用，并可供受损部位周围的细胞进入其中生长，促进受损组织愈合。
为实现上述目的，本发明采用以下技术方案：
一种制备具有局部止血或抗凝血功能的胶原材料的方法，包括以下步骤：
1 、通过直接与胶原中极性带正电荷的氨基酸发生化学反应，从而减少材料的正电荷，使材料带负电。
2 、通过直接与胶原中极性带负电荷的氨基酸发生化学反应，从而减少材料的负电荷，使材料带正电。
3 、在弱酸性至弱碱性条件下（pH6.0-8.0）和醛类参与下，或者在碱性条件下和环氧化合物A参与下，通过往胶原肽链中碱性氨基酸的氨基引入极性带电氨基酸基团，使材料带负电；这里的环氧化合物A至少带有2个或2个以上的环氧基；极性带电氨基酸基团可以带正电，也可以带负电。环氧基开环后分别与胶原肽链中碱性氨基酸的氨基以及引入的极性带电氨基酸的氨基发生反应，将极性氨基酸接入胶原基材。
4 、在环氧化合物参与和碱性催化剂作用下，通过往胶原肽链上酸性氨基酸的羧基引入极性带正电氨基酸基团，使材料带正电；这里的碱性催化剂可以是吡啶，异喹啉等化合物，浓度范围为0.1%-10%(v/v)。环氧化合物中的环氧基开环后在催化剂作用下与胶原肽链中酸性氨基酸的羧基与引入的极性带电氨基酸的羧基发生反应，将极性氨基酸接入胶原基材。
5 、通过活化胶原中极性带负电荷的氨基酸上的羧基，促使其与溶液中加入的游离氨基发生缩合反应，减少胶原基材上的负电荷，从而使材料带正电；这里的活化物可以是碳亚二胺类化合物，浓度范围为0.1%-5%（w/v），加入的游离氨基可以是极性氨基酸。
6 、按以上1-5中任选一种方案处理后，再对材料表面进行短时间化学处理，使材料表面电性与内部电性不同。这里的化学处理，主要是对1-5中反应形成的酯键进行破坏，使其水解成-COO - ，时间范围为5-30分钟。
较佳的，所述步骤1中所述化学反应为醛类或环氧类化合物与胶原中赖氨酸上的ε - 氨基反应；优选所述醛类为戊二醛，更优选戊二醛体积浓度为0.1%-2%；优选所述环氧类化合物为 乙二醇二缩水甘油醚和/或聚环氧丙烷。
较佳的，所述步骤1具体为： 在 pH6.0-8.0条件下，将胶原与戊二醛（体积浓度0.1%-2%）进行反应，反应过程如图2所示，反应时间范围为30分钟到14天；制成的胶原基材在溶液中带负电荷，具有抗凝血特性；例如，将牛心包置于加有pH 7.4 的PBS缓冲液的0.6%（v/v）戊二醛溶液中3天。或者在碱性条件下（pH8.0-10.5），将胶原与环氧化合物进行反应，环氧化合物中的环氧基与赖氨酸的ε - 氨基发生反应，反应过程如图3所示，反应时间范围为1小时到14天；反应完成后，胶原基材在溶液中带负电荷，具有抗凝血特性；例如，将牛心包置于加有碳酸钠 - 碳酸氢钠缓冲液的乙二醇二缩水甘油醚溶液中9天。
较佳的，步骤2中所述化学反应为乙二醇二缩水甘油醚在碱性催化剂作用下与胶原中极性带负电荷的氨基酸上的羧基发生反应；胶原与乙二醇二缩水甘油醚用量比例为1g胶原加入50mL，碱性催化剂为吡啶或异喹啉，浓度范围为0.1%-10%（v/v）。
较佳的，步骤2具体为：在0.5%（v/v）吡啶催化作用下，将胶原如牛心包与2%（v/v）乙二醇二缩水甘油醚按1g：50mL的比例进行反应。反应时间为1小时到14天。反应完成后，胶原基材在溶液中带正电荷，具有促进凝血的特性。
较佳的，步骤3中所述环氧化合物A为乙二醇二缩水甘油醚和/或聚环氧丙烷，所述极性带电氨基酸基团为赖氨酸、天冬氨酸和/或谷氨酸。
较佳的，所述步骤3具体为：在弱酸性至弱碱性条件下（Ph6.0-8.0），将胶原与戊二醛（体积浓度0.1%-2%）和赖氨酸（质量浓度1%）体积比为1:1的混合溶液发生反应，此处 胶原与混合溶液的用量比例为： 1g 胶原加混合溶液50mL， 反应过程如图4所示。反应时间范围为30分钟到14天；或者在碱性条件下pH8.0-10.5，将胶原与乙二醇二缩水甘油醚：赖氨酸的质量比为1:1的混合溶液进行反应，乙二醇二缩水甘油醚中的两个环氧基分别与胶原肽链中赖氨酸的ε - 氨基和溶液中赖氨酸的ε - 氨基发生反应，将溶液中的赖氨酸接入胶原基材，反应时间范围为1小时到14天。反应过程如图4所示。反应完成后，胶原基材在溶液中带负电荷，具有抗凝血特性。
较佳的，步骤4中所述极性带正电氨基酸基团为赖氨酸，所述环氧类化合物为乙二醇二缩水甘油醚和/或聚环氧丙烷。
较佳的，步骤4具体为：在0.5%（v/v）吡啶催化作用下，将胶原如牛心包浸入赖氨酸与乙二醇二缩水甘油醚比例为1:2( w/w) 的混合溶液中进行反应，反应过程如图4所示。反应时间为1小时到14天。反应完成后，胶原基材在溶液中带正电荷，具有促进凝血的特性。
较佳的，步骤5中所述极性氨基酸为赖氨酸。
较佳的，步骤5具体为：在酸性条件下（pH4.0-6.5），将胶原与EDC（碳化二亚胺）和NHS （ N- 羟基琥珀酰亚胺）的混合液（EDC：NHS质量比为5:1）发生反应，碳化二亚胺中的-N=C=N-基团与天冬氨酸和谷氨酸上的-COOH发生反应，形成不稳定的O-异酰基脲结构，NHS使其形成稳定的酯基，再与溶液中的游离氨基反应。反应过程如图5所示。反应完成后，整个胶原基材在溶液中带正电荷，具有止血特性。例如，将小牛皮置于含有 1%(w/w) 赖氨酸、pH 为5.5 - 6.7的MES缓冲液、碳化二亚胺：NHS比例为5:1（w/w）的溶液中1-3小时，碳化二亚胺浓度为1%(w/v)。
较佳的，步骤6具体为：在0.5%（v/v）吡啶催化作用下，将胶原如牛心包浸入赖氨酸与乙二醇二缩水甘油醚比例为1:2（w/w）的混合溶液中进行反应14天，然后置于弱酸性条件下（pH5.5-6.5）5-10分钟，使胶原基材上靠近接入的赖氨酸基团的酯键发生水解，使胶原基材表面恢复中性或略呈负电性，而基材内部依然带正电。
本发明所述胶原为：动物组织胶原，如牛心包，小牛皮。
本发明的有益效果在于：（1）未破坏天然胶原螺旋区结构，保留其生物活性，相较现有的胶原止血海绵，更有利于组织吸附和进入生长；（2）通过改变胶原基材电性，可使其具有促进凝血或者抗凝血作用，满足不同需求。
附图说明
图 1 中， a 为 Lys 结构式， b 为 Asp 结构式， c 为 Glu 结构式；
图 2 为 碱性催化剂作用下，胶原中天冬氨酸上的羧基与环氧化合物发生反应的过程；
图 3 为胶原中赖氨酸上的ε - 氨基与戊二醛发生反应的过程 ；
图 4 为碱性条件下，胶原中赖氨酸上的ε - 氨基与环氧化合物发生反应的过程 ；
图 5 为弱酸性至弱碱性条件下，戊二醛与胶原中赖氨酸上的ε - 氨基以及溶液中赖氨酸上的氨基发生反应的过程 ；
图 6 为碱性条件下，环氧化合物与胶原中赖氨酸上的ε - 氨基以及溶液中赖氨酸上的氨基发生反应的过程 ；
图 7 为碱性催化剂作用下，胶原中天冬氨酸上的羧基与环氧化合物以及加入的赖氨酸发生反应的过程；
图 8 为 胶原中天冬氨酸上的羧基 与碳化二亚胺 /NHS 溶液发生反应的过程。
具体实施方式
1 、在弱酸性至弱碱性条件下（pH6.0-8.0），将胶原与戊二醛（体积浓度0.1%-2%）发生反应， 此处胶原与戊二醛的用量比例为： 1g 胶原加1%（v/v）戊二醛50mL， 反应过程如图2所示。反应时间范围为30分钟到14天。制成的胶原基材在溶液中带负电荷，具有抗凝血特性。例如，将牛心包置于加有pH7.4的PBS缓冲液的0.6%（v/v）戊二醛溶液中3天。或者在碱性条件下（pH8.0-10.5）， 按 1g 胶原加2%（v/v）乙二醇二缩水甘油醚50mL的比例 将胶原与 乙二醇二缩水甘油醚 进行反应， 乙二醇二缩水甘油醚 中的环氧基与赖氨酸的ε - 氨基发生反应，反应过程如图3所示。反应时间范围为1小时到14天。反应完成后，胶原基材在溶液中带负电荷，具有抗凝血特性。例如，将牛心包置于加有pH8.0-10.5的碳酸钠 - 碳酸氢钠缓冲液的乙二醇二缩水甘油醚溶液中9天,此处，碳酸钠 - 碳酸氢钠 缓冲液为 0.04mol/L ，乙二醇二缩水甘油醚含量 0.02% （ v/v ） 。以上两种方法所得材料各取1cm2置于5mL加有柠檬酸钠的血浆中，再滴几滴1mol/L的CaCl2溶液，均于 8-9 分钟即开始出现凝血反应，正常凝血则需5-6分钟。
2 、在0.5%（v/v）吡啶催化作用下，将牛心包与2%（ v/v ） 乙二醇二缩水甘油醚按1g：50mL的比例进行反应。反应时间为1小时到14天。反应完成后，胶原基材在溶液中带正电荷，具有促进凝血的特性，所得材料取1cm2置于5mL加有柠檬酸钠的血浆中，再滴几滴1mol/L的CaCl2溶液，3-4分钟即开始出现凝血反应，正常凝血则需5-6分钟。
3 、在弱酸性至弱碱性条件下（ pH6.0-8.0 ），将胶原与戊二醛（体积浓度 0.1%-2% ）和赖氨酸（质量浓度 1% ）体积比为 1:1 的混合溶液发生反应，此处胶原与混合溶液的用量比例为： 1g 胶原加混合溶液 50mL ，反应过程如图 4 所示。反应时间范围为 30 分钟到 14 天；或者在碱性条件下如 pH8.0-10.5 的碳酸钠 - 碳酸氢钠缓冲液的乙二醇二缩水甘油醚， 此处，碳酸钠 - 碳酸氢钠 缓冲液为 0.04mol/L ，乙二醇二缩水甘油醚含量 0.02% （ v/v ），将胶原与乙二醇二缩水甘油醚：赖氨酸为 1:1 （ w/w ）的混合溶液进行反应，乙二醇二缩水甘油醚中的两个环氧基分别与胶原肽链中赖氨酸的ε - 氨基和溶液中赖氨酸的ε - 氨基发生反应，将溶液中的赖氨酸接入胶原基材，反应时间范围为 1 小时到 14 天。反应过程如图 5 所示。反应完成后，胶原基材在溶液中带负电荷，所得材料取 1cm2 置于 5mL 加有柠檬酸钠的血浆中，再滴几滴 1mol/L 的 CaCl2 溶液， 8-10 分钟后才开始出现凝血反应，正常凝血则需 5-6 分钟。
4 、在0.5%（ v/v ） 吡啶催化作用下，将牛心包浸入赖氨酸与乙二醇二缩水甘油醚比例为1:2（ w/w ） 的混合溶液中进行反应，反应过程如图6所示。反应时间为1小时到14天。反应完成后，胶原基材在溶液中带正电荷，具有促进凝血的特性，所得材料取1cm2置于5mL加有柠檬酸钠的血浆中，再滴几滴1mol/L的CaCl2溶液，3-4分钟即开始出现凝血反应，正常凝血则需5-6分钟。
5 、在酸性条件下（pH4.0-6.5），将胶原与EDC（碳化二亚胺）和NHS （ N- 羟基琥珀酰亚胺）的混合液发生反应，碳化二亚胺中的-N=C=N-基团与天冬氨酸和谷氨酸上的-COOH发生反应，形成不稳定的O-异酰基脲结构，NHS使其形成稳定的酯基，再与溶液中的游离氨基反应。反应过程如图7所示。反应完成后，整个胶原基材在溶液中带正电荷，具有止血特性。例如， 将小牛皮置于含有 1%(w/w) 赖氨酸、pH 为5.5 - 6.7的MES缓冲液、碳化二亚胺：NHS比例为5:1（ w/w ） 的溶液中1-3小时，碳化二亚胺浓度为1%（ w/w ） 。所得材料取1cm2置于5mL加有柠檬酸钠的血浆中，再滴几滴1mol/L的CaCl2溶液，2-3分钟即开始出现凝血反应，正常凝血则需5-6分钟。
6、在0.5%吡啶（v/v）催化作用下，将牛心包浸入赖氨酸与乙二醇二缩水甘油醚比例为1:2（w/w）的混合溶液中进行反应14天，然后置于pH为5.5 - 6.5的MES缓冲液5-10分钟，使胶原基材上靠近接入的赖氨酸基团的酯键发生水解，使胶原基材表面恢复中性或略呈负电性，而基材内部依然带正电。所得材料取1cm2置于5mL加有柠檬酸钠的血浆中，再滴几滴1mol/L的CaCl2溶液，将凝血反应后的产品做病理切片，观察得血块集中在胶原内部。

Claims (10)

  1. 一种制备具有止血或抗凝血功能的胶原材料的方法，包括以下步骤：
    （1）通过直接与胶原中极性带正电荷的氨基酸发生化学反应，从而减少材料的正电荷，使材料带负电；或
    （2）通过直接与胶原中极性带负电荷的氨基酸发生化学反应，从而减少材料的负电荷，使材料带正电；或
    （3）在pH6.0-8.0弱酸性至弱碱性条件下和醛类参与下，或者在碱性条件下和环氧化合物A参与下，通过往胶原肽链中碱性氨基酸的氨基引入极性带电氨基酸基团，使材料带负电，所述环氧化合物A至少带有2个环氧基，所述极性带电氨基酸基团带正电或带负电；或
    （4）在环氧化合物参与和碱性催化剂作用下，通过往胶原肽链上酸性氨基酸的羧基引入极性带正电氨基酸基团，使材料带正电；环氧化合物中的环氧基开环后在催化剂作用下与胶原肽链中酸性氨基酸的羧基与引入的极性带电氨基酸的羧基发生反应，将极性氨基酸接入胶原基材；或
    （5）通过使用活化物活化胶原中极性带负电荷的氨基酸上的羧基，促使其与溶液中加入的游离氨基发生缩合反应，减少胶原基材上的负电荷，从而使材料带正电；
    （6）按以上（1）-（5）中任选一种方案处理后，再对材料表面进行短时间化学处理，使材料表面电性与内部电性不同，所述化学处理，主要是对（1）-（5）中反应形成的酯键进行破坏，使其水解成-COO-，时间范围为5-30分钟。
  2. 根据权利要求1所述的方法，其中所述（1）中所述化学反应为醛类或环氧类化合物与胶原中赖氨酸上的ε-氨基反应。
  3. 根据权利要求1所述的方法，其中所述（3）中所述环氧化合物A为乙二醇二缩水甘油醚和/或聚环氧丙烷，所述极性带电氨基酸基团为赖氨酸、天冬氨酸和/或谷氨酸。
  4. 根据权利要求1所述的方法，其中所述（4）中所述极性带正电氨基酸基团为赖氨酸，所述环氧类化合物为乙二醇二缩水甘油醚和/或聚环氧丙烷，所述碱性催化剂是浓度范围为0.1%-10%(w/v)的吡啶和/或异喹啉等化合物。
  5. 根据权利要求1所述的方法，其中所述（5）中所述活化物是浓度范围为0.1%-5%(w/v)的碳亚二胺类化合物，所述加入的游离氨基是极性氨基酸。
  6. 根据权利要求2所述的方法，其中所述醛类为体积浓度为0.1%-2%的戊二醛；所述环氧类化合物为乙二醇二缩水甘油醚和/或聚环氧丙烷。
  7. 根据权利要求6所述的方法，其中（1）具体为：在 pH6.0-8.0条件下，将胶原与戊二醛进行反应，反应时间范围为30分钟到14天，制成的胶原基材在溶液中带负电荷，具有抗凝血特性；或者在pH8.0-10.5碱性条件下，将胶原与环氧化合物进行反应，反应时间范围为1小时到14天；反应完成后，胶原基材在溶液中带负电荷，具有抗凝血特性。
  8. 根据权利要求3所述的方法，其中（2）具体为：碱性条件下pH8.0-10.5，将胶原与乙二醇二缩水甘油醚：赖氨酸的质量比为1:1的混合溶液进行反应，反应时间范围为1小时到14天，反应完成后，胶原基材在溶液中带负电荷，具有抗凝血特性。
  9. 根据权利要求4所述的方法，其中（3）具体为：在0.5%（v/v）吡啶催化作用下，将胶原浸入赖氨酸与乙二醇二缩水甘油醚比例为1:2（w/w）的混合溶液中进行反应，反应时间为1小时到14天，反应完成后，胶原基材在溶液中带正电荷，具有促进凝血的特性。
  10. 根据权利要求5所述的方法，其中（4）具体为：在酸性条件下pH4.0-6.5，将胶原与EDC和NHS的混合液发生反应，EDC：NHS质量比为5:1，反应完成后，整个胶原基材在溶液中带正电荷，具有止血特性。
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