CN101323670B - Medical grade reconstructed collagen cross-linking modified method - Google Patents
Medical grade reconstructed collagen cross-linking modified method Download PDFInfo
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- CN101323670B CN101323670B CN200810045663XA CN200810045663A CN101323670B CN 101323670 B CN101323670 B CN 101323670B CN 200810045663X A CN200810045663X A CN 200810045663XA CN 200810045663 A CN200810045663 A CN 200810045663A CN 101323670 B CN101323670 B CN 101323670B
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Abstract
The invention discloses a method for cross-linking and modifying medical-grade reconstructed collagen, which is characterized in that 1 to 5 weight parts of phosphate solution with the concentration of 0.1 mol/L to 0.5mol/L is added into 5 to 25 weight parts of uniform regular collagen solution with the concentration of 5 mg/ml to 10mg/ml at the temperature of 15 DEG C to 20 DEG C while slowly stirring. Acid solution or aqueous alkali is used for adjusting the pH value of the mixed solution to 7.0 to 8.5. The mixed solution stands for 6 hours to 12 hours at the temperature of 25 DEG C to 40 DEG C and humidity of 50 percent to 70 percent so as to form uniform colloid. The colloid is sequentially arranged in the environment of minus 20 DEG C to minus 40 DEG C and minus 60 DEG C to minus 80 DEG C to be frozen for 24 hours to 48 hours, and then dehydrated in a lyophilizer to obtain the reconstructed collagen with cross-linked and modified ions.
Description
Technical field
The present invention relates to a kind of medical grade reconstructed collagen cross-linking modified method, belong to the bio-medical material preparation field.
Background technology
Collagen is vertebrate basic structure material, is the protein of Mammals in-vivo content maximum, also is the main protein that constitutes animal connective tissue.In vertebrates, account for about 1/3 of gross protein, mainly be present in bone, skin and the tendon.Type i collagen claims tropocollagen (tropocollagen) again, its molecule is clavate, be about 300nm, the about 1.5nm of diameter, molecular weight is about 300,000, and its basic structure is three strands of chains, form by two α 1 (I) and a α 2 (I) chain, each gang α chain contains 1056 amino-acid residues, and three strands of α chains are in the dextrorotation mode, mutually three bursts of superhelixs of coiled.In fact all dissimilar collagens all form in the mode of triple helix, and difference only is polypeptide fragment that the stage casing triple helix of each Collagen Type VI forms difference to some extent, thereby is folded into different three-D space structures.Up to the present, nearly 19 kinds of the collagen-type of having reported.
The collagen in any kind of system source, its amino acid is formed all a distinguished feature, and promptly glycine accounts for 1/3 of collagen amino acid residue, and proline(Pro) accounts for 1/4, and hydroxylysine and oxyproline are still arranged, and it is peculiar that this two seed amino acid belongs to tropocollagen molecule institute.Amino acid assortment order as for its polypeptide chain has certain rules, promptly repeats Gly-X-Y, and wherein the X position is a proline(Pro) mostly, and Y can be other amino acid, and oxyproline also more often occurs.
Collagen is compared with other biological macromolecular material such as Mierocrystalline cellulose, chitosan etc. as a kind of biomaterial, has following three aspect characteristics: the firstth, and mechanical property, its physical strength, viscous deformation and anti-fatigue ability are all placed in the middle in the character of biomaterial.The secondth, chemical property, water rate of permeation and water absorbability are all very high, and its oilness and stability are also fine.The 3rd is biology performance, and its biological activity is lower, nonirritant, non-toxic reaction behind the implantable bioartificial body.
Reconstructed collagen is to remove both sides end peptide, the soluble collagen of reconstructed fiber by biochemical reaction.Can be prepared into medical device or be used as other thorough fare as certain composition, its mechanical property is the mechanical property extreme difference of its hygrometric state particularly.There is cytotoxicity in common Chemical Crosslinking Methods, and the physical crosslinking method is difficult to its degraded of control.
Collagenic material is the good biomaterial that can be used for guide tissue regeneration of a class.Owing to have no antigen, good biocompatibility, can participate in organization healing process and degradation speed and advantage such as can regulate as required, it has important use to be worth at the aspects such as reparation of tendon, ligament, periodontal tissue and peritonaeum.
Collagen is content rich in protein the most in the human body protein, and can make it be transformed into portable prosthese by crosslinked method, so collagen plays an important role in organizational project.The heart valve bioprosthesis of making as crosslinked with collagen, artificial skin etc.Collagen is found the biology mechanism that psoriasis forms also through being usually used in making the model system of organoid as psoriasic skin model.
Having emerged a collection of in the world is the biological products of main component with the collagen protein, comprises soluble collagen, insoluble collagen and the matrix material of being made up of collagen and non-collagen stroma.These materials can be used for preparing collagen solution, collagen paste, collagen film, collagen suture etc., thereby are widely used in reparation, shaping and beauty, neurotization and the blood vessel valve surgery etc. of wound and burn.
Therefore medical collagen material and goods research, exploitation and listing along with the novel collagen medical device is understood and understanding by user and patient, have great market potential and development and application values.
Summary of the invention
The objective of the invention is defective, a kind of medical grade reconstructed collagen cross-linking modified method that provides at the prior art existence.Be characterized in a kind of ionic crosslinking method, the degraded of having avoided cytotoxicity that Chemical Crosslinking Methods may bring and physical crosslinking (energetic ray, high temperature) to cause, this method reconstructed collagen both no cytotoxicity material does not cause the reduction of the mechanical property of materials yet, and purity is high and be dissolved in acid or basic solution.
Purpose of the present invention is realized that by following technical measures wherein said raw material umber is parts by weight except that specified otherwise.
Reconstructed collagen cross-linking modified method may further comprise the steps:
With concentration is phosphate solution 1-5 part of 0.1-0.5mol/L, in temperature 15-20 ℃, slowly stirring and joining concentration is in the uniform common collagen solution 5-25 part of 5-10mg/ml, the pH value of regulating mixing solutions with acid or alkaline solution is 7.0-8.5, mixing solutions is 25-40 ℃ in temperature, humidity is 50%-70%, left standstill 6-12 hour, make it to become even colloid, colloid inserts successively-20--40 ℃ ,-environment of 60--80 ℃ in after freezing 24-48 hour, in the freeze drier dehydration, obtain the reconstructed collagen of ionomer modification.
Phosphoric acid salt is any in potassiumphosphate, dipotassium hydrogen phosphate, potassium primary phosphate, sodium phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, calcium phosphate and the secondary calcium phosphate.
Prepare as stated above cross-linking modified after the medical grade reconstructed collagen material.
Medical grade reconstructed collagen material after cross-linking modified is used for the organizational project body and is implanted into.
The performance test of cross-linking modified reconstructed collagen
Cross-linking modified reconstructed collagen sponge is implanted 16 weeks, 20 week and 35 all photos in subcutaneous rat, as shown in Figure 1, 2, 3, the result shows cross-linking modified reconstructed collagen no cytotoxicity, in the rat body, can keep not degrading for a long time, make up the implantation carrier that provides necessary for organizational project reparation and organ.
Cross-linking modified reconstructed collagen sponge is implanted 16 week and 20 all tissue slice photos in subcutaneous rat, and shown in Fig. 4,5, the result shows that cross-linking modified reconstructed collagen helps the adhesion growth of cell and growing into of blood vessel.
Compared with the prior art the present invention has the following advantages:
1, no cytotoxicity
2, control degree of crosslinking easily, further control degradation speed in vivo can make the collagenic material degradation time in vivo after cross-linking modified be controlled at for 12 to 78 weeks;
3. be easy to moulding, can obtain the materials such as collagen powder, collagem membrane and collagen sponge after cross-linking modified easily.
4. improved the mechanical property of collagenic material hygrometric state
Description of drawings
Fig. 1 implants 16 all photo figure for cross-linking modified back collagen sponge in subcutaneous rat
Fig. 2 implants 20 all photo figure for cross-linking modified back collagen sponge in subcutaneous rat
Fig. 3 implants 35 all photo figure for cross-linking modified back collagen sponge in subcutaneous rat
Fig. 4 implants 16 all tissue slice figure for cross-linking modified back collagen sponge in subcutaneous rat
Fig. 5 implants 20 all tissue slice figure for cross-linking modified back collagen sponge in subcutaneous rat
Embodiment
Below by embodiment the present invention is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified; can not be interpreted as limiting the scope of the invention, some nonessential improvement and adjustment that the person skilled in the art in this field can make the present invention according to the content of the invention described above.
Embodiment 1
With concentration is the potassium phosphate solution 1ml of 0.1mol/L, in 15 ℃ of temperature, slowly stirring and joining concentration is among the uniform common collagen solution 5ml of 5mg/ml, and the pH value of regulating mixing solutions with acid or alkaline solution is 7.0, and mixing solutions is 25 ℃ in temperature, humidity is 50%, left standstill 6 hours, and made it to become even colloid, colloid is inserted in-20 ℃ ,-60 ℃ the environment after freezing 24 hours successively, in the freeze drier dehydration, obtain the reconstructed collagen of ionomer modification.
Embodiment 2
With concentration is the sodium radio-phosphate,P-32 solution 5ml of 0.5mol/L, in 20 ℃ of temperature, slowly stirring and joining concentration is among the uniform common collagen solution 25ml of 10mg/ml, and the pH value of regulating mixing solutions with acid or alkaline solution is 8.5, and mixing solutions is 40 ℃ in temperature, humidity is 70%, left standstill 12 hours, and made it to become even colloid, colloid is inserted in-40 ℃ ,-80 ℃ the environment after freezing 48 hours successively, in the freeze drier dehydration, obtain the reconstructed collagen of ionomer modification.
Embodiment 3
With concentration is the dipotassium hydrogen phosphate solution 2ml of 0.2mol/L, in 16 ℃ of temperature, slowly stirring and joining concentration is among the uniform common collagen solution 10ml of 6mg/ml, and the pH value of regulating mixing solutions with acid or alkaline solution is 7.2, and mixing solutions is 28 ℃ in temperature, humidity is 55%, left standstill 8 hours, and made it to become even colloid, colloid is inserted in-25 ℃ ,-65 ℃ the environment after freezing 26 hours successively, in the freeze drier dehydration, obtain the reconstructed collagen of ionomer modification.
Embodiment 4
With concentration is the potassium dihydrogen phosphate 3ml of 0.3mol/L, in 17 ℃ of temperature, slowly stirring and joining concentration is among the uniform common collagen solution 15ml of 7mg/ml, and the pH value of regulating mixing solutions with acid or alkaline solution is 7.4, and mixing solutions is 30 ℃ in temperature, humidity is 60%, left standstill 9 hours, and made it to become even colloid, colloid is inserted in-30 ℃ ,-70 ℃ the environment after freezing 30 hours successively, in the freeze drier dehydration, obtain the reconstructed collagen of ionomer modification.
Embodiment 5
With concentration is the calcium phosphate solution 4ml of 0.4mol/L, in 18 ℃ of temperature, slowly stirring and joining concentration is among the uniform common collagen solution 20ml of 8mg/ml, and the pH value of regulating mixing solutions with acid or alkaline solution is 7.6, and mixing solutions is 32 ℃ in temperature, humidity is 65%, left standstill 10 hours, and made it to become even colloid, colloid is inserted in-35 ℃ ,-75 ℃ the environment after freezing 35 hours successively, in the freeze drier dehydration, obtain the reconstructed collagen of ionomer modification.
Embodiment 6
With concentration is the disodium phosphate soln 5ml of 0.5mol/L, in 19 ℃ of temperature, slowly stirring and joining concentration is among the uniform common collagen solution 25ml of 9mg/ml, and the pH value of regulating mixing solutions with acid or alkaline solution is 8, and mixing solutions is 34 ℃ in temperature, humidity is 68%, left standstill 11 hours, and made it to become even colloid, colloid is inserted in-40 ℃ ,-80 ℃ the environment after freezing 40 hours successively, in the freeze drier dehydration, obtain the reconstructed collagen of ionomer modification.
Claims (3)
1. medical grade reconstructed collagen cross-linking modified method, it is characterized in that this method is is the phosphate solution 1-5 weight part of 0.1-0.5mol/L with concentration, in temperature 15-20 ℃, slowly stirring and joining concentration is in the uniform common collagen solution 5-25 weight part of 5-10mg/ml, the pH value of regulating mixing solutions with acid or alkaline solution is 7.0-8.5, mixing solutions is 25-40 ℃ in temperature, humidity is 50%-70%, left standstill 6-12 hour, make it to become even colloid, colloid inserts successively-20--40 ℃, after freezing 24-48 hour,, obtain the reconstructed collagen of ionomer modification in-60--80 ℃ the environment in the freeze drier dehydration.
2. medical grade reconstructed collagen cross-linking modified according to claim 1 method is characterized in that phosphoric acid salt is any in potassiumphosphate, dipotassium hydrogen phosphate, potassium primary phosphate, sodium phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, calcium phosphate and the secondary calcium phosphate.
According to claim 1 method prepare cross-linking modified after the medical grade reconstructed collagen material.
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| CN200810045663XA CN101323670B (en) | 2008-07-29 | 2008-07-29 | Medical grade reconstructed collagen cross-linking modified method |
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