CN102258807B - Porcine acellular dermal matrix (PADM) pore adjusting method for tissue engineering - Google Patents
Porcine acellular dermal matrix (PADM) pore adjusting method for tissue engineering Download PDFInfo
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- CN102258807B CN102258807B CN 201110199312 CN201110199312A CN102258807B CN 102258807 B CN102258807 B CN 102258807B CN 201110199312 CN201110199312 CN 201110199312 CN 201110199312 A CN201110199312 A CN 201110199312A CN 102258807 B CN102258807 B CN 102258807B
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- dermal matrix
- acellular dermal
- biomacromolecule
- pig acellular
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Abstract
The invention discloses a porcine acellular dermal matrix (PADM) pore adjusting method for tissue engineering, which is implemented by adding anion biomacromolecules or cation biomacromolecules into a sufficiently wetted PADM water solution and regulating the electrokinetic potential (zeta potential) of collagen, wherein the anion biomacromolecules are sodium polyaspartate, the cation biomacromolecules are water-soluble chitosan, and the weight ratio of the biomacromolecules to the PADM is 1:5-1:25. The method disclosed by the invention effectively adjusts the pore size of the PADM, and adjusts the structure pattern of the collagen beam; and the obtained PADM has the natural structure of collagen, which predicts that the material can have wide application prospects in repair medicine.
Description
Technical field
The present invention relates to the accent hole method of a kind of used in tissue engineering pig acellular dermal matrix (PADM), relate in particular to a kind of method, belong to technical field of biological material with biomacromolecule regulation and control used in tissue engineering pig acellular dermal matrix (PADM) aperture.
Background technology
The development of skin and bone renovating material is the direction that the large quantities of scientists of field of tissue engineering technology make great efforts always.Autotransplantation is occupied an leading position always clinically.But limited from body bone source, and the danger that heteroplastic transplantation has immunologic rejection and catches.People have developed various materials and have been used for skin and bone tissue engineer field.But these materials respectively have deficiency, can not reach requirement clinically fully.Because the organic principle in skin and the bone all mainly is to be made of type i collagen, so collagen base timbering material has just become the research focus.But traditional collagen source is for extracting from the tendon of Mus tail and cattle, horse, and leaching process has not only destroyed the natural structure of collagen, has reduced its biocompatibility and mechanical performance, and the extraction cost height.
Material with The Nomenclature Composition and Structure of Complexes similar with basic stitch of tissue engineering bracket material principle prompting will have more excellent biology performance.
Pig acellular dermal matrix (PADM) mainly is made of type i collagen, and organic composition is quite similar in composition and nature bone and the skin.Its extraction and purification process are comparatively ripe, that the existing portioned product of the U.S. is authenticated and is applied to by FDA is clinical (Permacol, Covedien).And its research on bone tissue engineer also begins to cause researchers' attention.As tissue engineering bracket material, its aperture size is extremely important, and it is related to the growing into and the transportation of metabolite and nutrient substance of migration, tissue of cell.The traditional control PADM hole shape looks and the method for size mainly contain chemical cross-linking agent cross-linking method, mechanical punching method and laser port-creating method.These methods are all having damage or are changing its natural structure PADM in varying degrees, and therefore, the accent hole method of developing new, more practical used in tissue engineering pig acellular dermal matrix (PADM) is imperative.
Summary of the invention
At the deficiency that exists in the existing P ADM aperture control method, the present invention seeks to propose a kind of method with biomacromolecule regulation and control used in tissue engineering pig acellular dermal matrix (PADM) hole dimension.
The accent hole method of used in tissue engineering pig acellular dermal matrix of the present invention (PADM), being that anion biomacromolecule or cation biomacromolecule are added in the pig acellular dermal matrix aqueous solution (pH=7) of abundant moistening, is that the zeta current potential is realized by the electric potential of regulating collagen; It is characterized in that: described anion biomacromolecule is a poly-aspartic-acid sodium, and described cation biomacromolecule is a water-soluble chitosan; The part by weight of described biomacromolecule and pig acellular dermal matrix is 1: 5～1: 25.
Wherein: described poly-aspartic-acid sodium selects molecular weight 2000-3500 dalton, density 1.1g/cm
3(20 ℃), pH (1%) are the poly-aspartic-acid sodium of 4-7; It is 85-87% that described water-soluble chitosan selects deacetylation, and viscosity (1%) is 30-300mpa.s, and pH (1%) is the water-soluble chitosan of 4-7.
The accent hole method of above-mentioned used in tissue engineering pig acellular dermal matrix, further concrete implementation step is:
(1) takes by weighing a certain amount of pig acellular dermal matrix, be that 1: 100～1: 200 part by weight mixes by pig acellular dermal matrix and ultra-pure water then, then in the constant temperature shaking table with 80-150 rev/min speed, with the abundant moistening 12-24 of pig acellular dermal matrix support hour;
(2) be that 1: 5～1: 25 ratio is dissolved in anion biomacromolecule or cation biomacromolecule in the above-mentioned water that contains support in the weight ratio of biomacromolecule and pig acellular dermal matrix, continue in the constant temperature shaking table speed responsing 12-24 hour with 80-150 rev/min;
(3) take out support, the shaking table that with deionized water and with speed is 80-130 rev/min was to its washing 3-5 time, each 30-120 minute;
(4) under subzero 70 ℃ of conditions, carry out freezing to the support after the washing, at subzero 50-60 ℃ of following lyophilization 12-16 hour, getting the aperture was the thick pig acellular dermal matrix support for the lamellar of 5-10 micron or loose collagen bundle structure of 50-300 micron, hole wall then.
In the accent hole method of above-mentioned used in tissue engineering pig acellular dermal matrix: pig acellular dermal matrix (PADM) is to adopt Chinese patent 031390633 described method preparation.
In the accent hole method of above-mentioned used in tissue engineering pig acellular dermal matrix: preferred 1: 120～1: 170 of the weight ratio of described pig acellular dermal matrix and ultra-pure water.
In the accent hole method of above-mentioned used in tissue engineering pig acellular dermal matrix: preferred 1: 7～1: 15 of the weight ratio of described biomacromolecule and pig acellular dermal matrix.
In the accent hole method of above-mentioned used in tissue engineering pig acellular dermal matrix: described anion biomacromolecule is a poly-aspartic-acid sodium, and described cation biomacromolecule is a water-soluble chitosan.
In the accent hole method of above-mentioned used in tissue engineering pig acellular dermal matrix: the preferred 90-120 of shaking table speed rev/min of described when washing, wash time is 45-90 minute at every turn, this step promptly will guarantee to remove the relict macromole, prevents that again support is subjected to mechanical damage.
The present invention has proposed the accent hole method of a kind of used in tissue engineering pig acellular dermal matrix (PADM) through experiment sieving, by selecting good biocompatibility, regulate the electric potential of pig acellular dermal matrix (PADM) tropocollagen molecule with the biomacromolecule (as poly-aspartic-acid sodium or water-soluble chitosan) of different electric charges, thereby regulate the contraction of collagen bundle, swelling reaches the purpose in control aperture, obtain having the skin and the osseous tissue collagen scaffold of excellent structure and composition, experiment confirm: pig acellular dermal matrix support of the present invention aperture is the 50-300 micron, hole wall is thick to be the lamellar or the loose collagen bundle structure of 5-10 micron, and described support aperture, the hole wall large profits is adjustable with this method, makes PADM that more wide application prospect be arranged on organizational project.In a word, method of the present invention is not only effectively regulated the aperture size of pig acellular dermal matrix, and the structure and morphology of adjustment collagen bundle, the pig acellular dermal matrix that obtains has kept the natural structure of collagen, indicates that this class material will have important clinical application value and social benefit in repairing medical science.
Description of drawings
The PADM porous support of Fig. 1 chitosan adjustment aperture 100-150 micron.
The PAMD porous support of Fig. 2 chitosan adjustment aperture 150-300 micron.
The PADM porous support of Fig. 3 poly-aspartic-acid sodium adjustment aperture 50-100 micron.
The PADM porous support of Fig. 4 poly-aspartic-acid sodium adjustment aperture 100-250 micron.
The specific embodiment
Embodiment 1: the aperture is in the preparation of the natural support of the PADM of 100-150 micron.
(1) take by weighing a certain amount of PADM, then it immersed in the ultra-pure water of 120 times of its weight, in the constant temperature shaking table with 80 rev/mins speed, with the abundant moistening of support 15 hours.
(2) accurately the chitosan of its weight 7% of weighing is dissolved in the water that contains support, and continuation with 120 rev/mins speed, was reacted 12 hours in the constant temperature shaking table.
(3) take out support, use deionized water wash 3 times, 60 minutes/time, shaking table speed was 80 rev/mins during washing.
(4) carry out under subzero 70 ℃ freezing, at last subzero 60 ℃ of lyophilizations 16 hours.
The PADM aperture that obtains after above-mentioned steps is at the 100-150 micron, and hole wall is thick to be the used in tissue engineering PADM of 5-10 micrometer fibers Shu Zucheng.
Embodiment 2: the aperture is in the preparation of the natural support of the PADM of 150-300 micron.
With embodiment 1, the quality of water-soluble chitosan is 20% of a PADM weight in the step (2);
Wash time is 5 times in the step (3).
The PADM aperture that obtains after above-mentioned steps is at the 150-300 micron, the used in tissue engineering PADM that hole wall is made up of the loose collagen sheet of 5-10 micron.
Embodiment 3: the aperture is in the preparation of the natural support of the PADM of 50-100 micron.
With embodiment 1, change water-soluble chitosan into poly-aspartic-acid sodium;
The quality of poly-aspartic-acid sodium is 8% of a PADM weight in the step (2);
The PADM aperture that obtains after above-mentioned steps is at the 50-100 micron, and hole wall is by the thick used in tissue engineering PADM support of forming for the lamellar collagen of 30-50 micron.
Embodiment 4: the aperture is in the preparation of the natural support of the PADM of 100-250 micron.
With embodiment 1, change water-soluble chitosan into poly-aspartic-acid sodium;
The quality of poly-aspartic-acid sodium is 20% of a PADM weight in the step (2);
Wash time is 5 times in the step (3).
The PADM aperture that obtains after above-mentioned steps is at the 100-250 micron, and hole wall is the used in tissue engineering PADM that the loose collagenous fiber bundle of 10-30 micron is formed.
Claims (8)
1. the accent hole method of a used in tissue engineering pig acellular dermal matrix (PADM), being that anion biomacromolecule or cation biomacromolecule are added in the pig acellular dermal matrix aqueous solution of abundant moistening, is that the zeta current potential is realized by the electric potential of regulating collagen; It is characterized in that: described anion biomacromolecule is a poly-aspartic-acid sodium, and described cation biomacromolecule is a water-soluble chitosan; The part by weight of described biomacromolecule and pig acellular dermal matrix is 1:5～1:25.
2. the accent hole method of used in tissue engineering pig acellular dermal matrix according to claim 1, it is characterized in that: described poly-aspartic-acid sodium selects molecular weight 2000-3500 dalton, density 1.1g/cm
3(20 ℃), pH are the poly-aspartic-acid sodium of 4-7.
3. the accent hole method of used in tissue engineering pig acellular dermal matrix according to claim 1, it is characterized in that: it is 85-87% that described water-soluble chitosan selects deacetylation, and viscosity is 30-300mpa.s, and pH is the water-soluble chitosan of 4-7.
4. the accent hole method of used in tissue engineering pig acellular dermal matrix according to claim 1 the steps include:
(1) takes by weighing a certain amount of pig acellular dermal matrix, be that the part by weight of 1:100～1:200 mixes by pig acellular dermal matrix and ultra-pure water then, then in the constant temperature shaking table with 80-150 rev/min speed, with the abundant moistening 12-24 of pig acellular dermal matrix support hour;
(2) be that the ratio of 1:5～1:25 is dissolved in anion biomacromolecule or cation biomacromolecule in the above-mentioned water that contains support in the weight ratio of biomacromolecule and pig acellular dermal matrix, continue in the constant temperature shaking table speed responsing 12-24 hour with 80-150 rev/min;
(3) take out support, the shaking table that with deionized water and with speed is 80-130 rev/min was to its washing 3-5 time, each 30-120 minute;
(4) under subzero 70 ℃ of conditions, carry out freezing to the support after the washing, at subzero 50-60 ℃ of following lyophilization 12-16 hour, getting the aperture was the thick pig acellular dermal matrix support for the lamellar of 5-10 micron or loose collagen bundle structure of 50-300 micron, hole wall then.
5. as the accent hole method of used in tissue engineering pig acellular dermal matrix as described in the claim 4, it is characterized in that: the weight ratio of described pig acellular dermal matrix and ultra-pure water is selected 1:120～1:170.
6. as the accent hole method of used in tissue engineering pig acellular dermal matrix as described in the claim 4, it is characterized in that: the weight ratio of described biomacromolecule and pig acellular dermal matrix is 1:7～1:15.
7. as the accent hole method of used in tissue engineering pig acellular dermal matrix as described in the claim 4, it is characterized in that: described anion biomacromolecule is a poly-aspartic-acid sodium, and described cation biomacromolecule is a water-soluble chitosan.
8. as the accent hole method of used in tissue engineering pig acellular dermal matrix as described in the claim 4, it is characterized in that: the shaking table speed during described washing is selected 90-120 rev/min, and wash time is 45-90 minute at every turn.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6281256B1 (en) * | 1997-03-31 | 2001-08-28 | The Regents Of The University Of Michigan | Open pore biodegradable matrices |
| WO2001087575A2 (en) * | 2000-05-12 | 2001-11-22 | The Regents Of The University Of Michigan | Reverse fabrication of porous materials |
| CN101417145A (en) * | 2008-12-04 | 2009-04-29 | 山东大学 | Bracket material for bone tissue engineer and preparation method thereof |
| CN102188751A (en) * | 2010-03-19 | 2011-09-21 | 温州医学院附属第一医院 | Laser micropore porcine acellular dermal matrix and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6281256B1 (en) * | 1997-03-31 | 2001-08-28 | The Regents Of The University Of Michigan | Open pore biodegradable matrices |
| WO2001087575A2 (en) * | 2000-05-12 | 2001-11-22 | The Regents Of The University Of Michigan | Reverse fabrication of porous materials |
| CN101417145A (en) * | 2008-12-04 | 2009-04-29 | 山东大学 | Bracket material for bone tissue engineer and preparation method thereof |
| CN102188751A (en) * | 2010-03-19 | 2011-09-21 | 温州医学院附属第一医院 | Laser micropore porcine acellular dermal matrix and preparation method thereof |
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