CN1546182A - Tissue engineering compound material for repairing nerve injury and its preparation - Google Patents
Tissue engineering compound material for repairing nerve injury and its preparation Download PDFInfo
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- CN1546182A CN1546182A CNA2003101189949A CN200310118994A CN1546182A CN 1546182 A CN1546182 A CN 1546182A CN A2003101189949 A CNA2003101189949 A CN A2003101189949A CN 200310118994 A CN200310118994 A CN 200310118994A CN 1546182 A CN1546182 A CN 1546182A
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- nerve injury
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Abstract
The invention discloses a complex type tissue engineering nerve damage renovation material, which comprises type I collagen, heparitin sulfate, type IV collagen and gelatin. The material has simple adjusting inner diameter and uniform single orientation axial bore, it is easy to be made into different external forms, e.g. cylinder or rectangle.
Description
Technical field
The present invention is intended to develop a kind of the have maincenter of good biocompatibility and biodegradability and the reparation bridge material behind the peripheral nerve injury, relate to multiple biological raw materials such as collagen, gelatin and aminoglycan with and processing technique and manufacture method.This material both can be used for the basic research that nerve injury is repaired, and also can be used for clinical human body spinal cord, the damage of peripheral nervous segmental, the bridge joint reparation after damaged.
Background technology
Extracellular matrix (ECM) is the basis that cell function and biological property were depended on and kept to cell for existence, is that the underlying carrier of life provides its support in order to keep structure and the place of biological function for biology.Functions such as the cell adhesion that ECM had, intercellular signal transmission, migration be unique, other materials are not available, and the function of organism is had far-reaching influence.ECM comprises three major types: collagen, Dan Baijutang, glycoprotein.Wherein collagen is the widest in the human body distribution, and accounting for 30%, one Collagen Type VI then is that content is maximum in the various collagens, and it is provided for keeping the support of structure for biological cell.Type i collagen is made up of three α chains, and constituting diameter is the fibril of the triple-helix structure of 67nm, is the key component of basic mode, has resistance to compression and keeps the function of space structure, mainly is distributed in the bone cornea, and tendon etc. are located; Gelatin is a kind of mixtures of polypeptides that is made by the tropocollagen molecule degraded, has 18 seed amino acids and has formed the gelatin peptide chain, and collagen extensively is present in bone, tendon and the connective tissue of animal, can be by bio-enzyme degradation.A large amount of experiments show: nervous tissue's engineering material made from extracellular matrix not only has excellent biological compatibility, also helps the delay and the growth of neuranagenesis fiber simultaneously.But, be unfavorable for spinal cord or peripheral nervous segmental damage regenerated fiber oriented growth, thereby the effect that has influenced treatment or tested because previously equal none set pattern of compound bridge joint carrier material internal structure is restrained (on pore size and direction).Preparation has the collagen bridge material in Y-direction aperture, does not still have similar report at present in the world, the internal void size that material that we develop has a material under the identical conditions evenly, hole is parallel---advantages such as complete and axially parallel.Therefore, in neural repair tissue engineering, have more suitable spinal cord and peripheral nervous characteristics, obtain spinal cord and peripheroneural effective reparation, regeneration along axial hole oriented growth and migration.In clinical and experiment, have a extensive future, act on huge.
Summary of the invention
Purpose of the present invention is intended to provide better compound tissue engineering nerve injury repairing material of a kind of biocompatibility mechanical property and preparation method thereof by the improvement to raw-material prescription and processing technique thereof, the not only inner microtubule type pore structure of this material with arrangement regulation, and pore size and direction can be regulated and control as required, help the growth of neuranagenesis fiber, can be used for spinal cord, Repair of Peripheral Nerve Injury; By adjusting each basis of preparation material, make this material have the effect of better promotion neuranagenesis fiber growth.
To achieve these goals, the technical solution adopted in the present invention: this material is mixed and made into collagen and aminoglycan, and material adopts type i collagen albumen (collagen I), IV collagen type (collagenIV), heparitin sulfate (heparan sulfate), gelatin (gelatin).Its formula proportion is: (230: 5: 1: 115)～(4600: 100: 1: 2300)
Above-mentioned prescription by adjusting the stranguria of cold type speed and the thermograde of raw mix, reaches preparation inside and have the evenly distributed micro-tubular structure of axially parallel, and the microtubule diameter has special nerve injury, the impairment renovation material of controllability under the thermograde condition; Its manufacture method is carried out according to the following steps:
1) takes by weighing a certain amount of type i collagen albumen by ratio and put into 0.05M acetum dissolving 24 hours by the concentration of 27.5mg/ml;
2) in 4 ℃ of isoperibols, 18000r/min stirs 90min, makes suspension;
3) other takes by weighing in a certain amount of heparitin sulfate, the IV collagen type adding 0.05M acetum by ratio and dissolved 24 hours;
4) at 4 ℃ of constant temperature, 18000r/min stirs 90min and makes suspension;
5) mix two kinds of suspensions and keep 4 ℃ of constant temperature, 18000r/min stirs 90min, makes the suspension of collagen protein and heparitin sulfate, and evacuation left standstill 12 hours.
6) the bridge material suspension for preparing being injected internal diameter is that 3mm, external diameter are the silica gel tube sealing both ends of 4.2mm, long 3cm to 20cm, slowly puts into the agent of profound hypothermia stranguria of cold type along tube axial direction, and admission velocity is controlled to be 10 * 10
-5Ms
-1To 10 * 10
-7Ms
-1
7) suspension-silica gel tube frozen material is put into the good aluminum dish of pre-cooling, lyophilizing 48h in-30 ℃, 100mtorr condition;
8) be warming up to 0 ℃ under the vacuum state and keep 24h, continue to be warming up to 22 ℃ again and keep 45 ~ 90min, remove vacuum, rise to room temperature;
9) material is soaked with 20% glutaraldehyde solidified in 24 hours, distilled water is dialysed repeatedly, crosslinked 16 hours again through ultraviolet radiation;
10) with the material aseptic sealed packages and place 2000 roentgen CO
60Illumination-based disinfection is 24 hours in the environment.
1. the novel spinal cord that the present invention developed, peripheral nerve reparation bridge material have, and the outer surface of material is full-closed structure.2. the microtubule pore size of material can be controlled in 20 μ m～300 μ m; 3. the direction of travel of microtubule is axial and parallel, even.In biological organization material was compound, the attached wall of wide-aperture material pair cell was creeped or is poured into as Olfactory essheathing cell, schwann cell etc. and play the support carrier function; The material of small-bore then is suitable for compound various nerve growth factor, medicine etc., is used for the bridge joint at spinal cord and the damaged place of peripheral nervous.The material surface of being developed does not have porous dehiscence, makes can prevent that the hypertrophy connective fiber from growing into damaged section implanting spinal cord or the damaged place of peripheral nervous, avoids hindering the oriented growth of neuranagenesis aixs cylinder.
The compound tissue engineering nerve injury repairing material that the present invention proposes has following characteristics:
1. adopted the improved biocompatible raw material, the internal milieu of more approaching biology helps neural regeneration more.Wherein IV collagen type (collagen IV) is the formation component of neural basement membrane, and heparitin sulfate (heparan sulfate) helps neural regeneration, and gelatin (gelatin) then is the optimal carrier of neurotrophic factor.
2. verified the optimum mixture ratio example of type i collagen albumen (collagen I), IV collagen type (collagen IV), heparitin sulfate (heparan sulfate), gelatin biomaterials such as (gelatin) and to the influence of material at aspects such as biological property and mechanical properties;
3. verified between temperature, speed and novel nervous tissue engineering repair materials internal structure and concerned;
4. verified the influence of various composition different proportions mixing to the material internal structure.
5. verify glue joins between the component, solidified optimum condition has increased material tensile strength and rigidity, better improved Mechanical Properties of Materials.
6. by the composition component ratio of control material, effectively reduced material decomposition rate in vivo.
7. developed 1. multistage adjustable speed adjusting and control instrument, made in the material processing stranguria of cold type speed 10 * 10
-5～1.5 * 10
-7Can arbitrarily carry out the regulation and control of multistage variable formula between the m/s; 2. the stranguria of cold type temperature controller makes the regulation and control arbitrarily between 0 ℃～-180 ℃ of stranguria of cold type temperature.
Compound tissue engineering nerve injury repairing material of the present invention is that a kind of novel spinal cord, peripheral nerve are repaired bridge material, both can directly implant spinal cord, peripheral nerve defection place; Also can be used as the carrier that each seed cell of nervous tissue's engineering is transplanted, can be compound, the medicine that carries various promotion neuranagenesis, be used for human body spinal cord, the damage of peripheral nervous segmental, the bridge joint after damaged; Can be used for the basic research of spinal cord, peripheral nervous sections injury repairing equally.
Description of drawings
Fig. 1 is the picture that axial tangent plane is observed under 400 times of scanning electron microscopies, and its material internal is the vertical micropore that moves towards parallel, aperture homogeneous, arrangement regulation;
Fig. 2 is the picture that axial tangent plane is observed under 70 times of Electronic Speculum, and display material inside is vertical microcellular structure.
Fig. 3 is the pictures of 45 ° of oblique sections under 300 times of Electronic Speculum, and display material inside is three-dimensional vertically tubular structure;
Fig. 4 is a picture under the 400 times of Electronic Speculum in cross section, and display material internal capillary internal diameter is homogeneous comparatively.
Concrete embodiment
Below in conjunction with processing technology and embodiment the present invention is described in further details.
According to technical scheme of the present invention, this compound tissue engineering nerve injury repairing material is mixed and made into collagen and aminoglycan, and material adopts type i collagen albumen (collagen I), IV collagen type (collagen IV), heparitin sulfate (heparan sulfate), gelatin (gelatin).Its formula proportion is: (230: 5: 1: 115)～(4600: 100: 1: 2300)
Above-mentioned material is in certain cryogenic temperature gradient environment, the thermograde rate of regression is the key factor of decision material internal microcellular structure pore size, micropore orientation, change and to be marked with speed that collagen-aminoglycan mixture silica gel tube enters the stranguria of cold type agent and can to reach material to required preparation pore size and hole direction and uniformity coefficient on internal structure and all have Modulatory character, thereby obtain compound spinal cord, peripheral nerve tissue's engineering injury repairing material.
Embodiment 1 (according to type i collagen albumen: IV collagen protein: heparitin sulfate: the ratio of gelatin is 2300: 50: 1: 1150):
According to technical scheme of the present invention, be example with collagen protein, gelatin and heparitin sulfate, its manufacture method is carried out according to the following steps:
1. take by weighing a certain amount of type i collagen albumen by ratio and put into 0.05M acetum dissolving 24 hours by the concentration of 27.5mg/ml;
2. in 4 ℃ of isoperibols, 18000r/min stirs 90min, makes suspension;
3. take by weighing in a certain amount of heparitin sulfate, the IV collagen type adding 0.05M acetum by ratio in addition and dissolved 24 hours;
4. at 4 ℃ of constant temperature, 18000r/min stirs 90min and makes suspension;
5. mix two kinds of suspensions and keep 4 ℃ of constant temperature, 18000r/min stirs 90min, makes the suspension of collagen protein and heparitin sulfate, and evacuation leaves standstill 12h.
6. the bridge material suspension for preparing being injected internal diameter is that 3mm, external diameter are the silica gel tube sealing both ends of 4.2mm, long 3cm to 20cm, slowly puts into the agent of profound hypothermia stranguria of cold type along tube axial direction, and admission velocity is controlled to be 10 * 10
-5Ms
-1, 5 * 10
-5Ms
-1To 10 * 10
-7Ms
-1
7. suspension-silica gel tube frozen material is put into the good aluminum dish of pre-cooling, lyophilizing 48h in-30 ℃, 100mtorr condition;
8. be warming up to 0 ℃ under the vacuum state and keep 24h, continue to be warming up to 22 ℃ again and keep 45 ~ 90min, remove vacuum, rise to room temperature;
9. material is soaked with 20% glutaraldehyde and solidified in 24 hours, distilled water is dialysed repeatedly, crosslinked 16 hours again through ultraviolet radiation;
10. with the material aseptic sealed packages and place 2000 roentgen CO
60Illumination-based disinfection is 24 hours in the environment.
Temperature and stranguria of cold type speed be the preparation this material two key factors.In order to obtain uniform and stable stranguria of cold type speed, grope by repeatedly testing, the applicant has developed multistage variable formula velometer, and stranguria of cold type speed and temperature all can be regulated and control within the required range.
The bridge material that the present invention developed has following characteristics: 1. the outer surface of material is a full-closed structure, and no ceasma can effectively stop scar tissue to be grown into; 2. the micro-pore diameter of material can artificially simply be regulated and control, and has both helped growing into of neuranagenesis fiber, helps the implantation of neural factor and seed cell again; 3. inner microtubule size is even, and arrangement regulation helps the oriented growth of neuranagenesis fiber; 4. zoopery proves this material good biocompatibility and the moderate neuranagenesis that helps of degradation speed; 5. after this material through special technology was handled, mechanical property was good, and tension and non-deformability are good.
Following example 2～8 each raw-material mixing ratio are identical.
Embodiment 2: adopt collagen (I type), heparitin sulfate and gelatin as raw material, its weight is respectively 1150mg, 0.5mg, 575mg, and implementation process is with example 1;
Embodiment 3: adopt collagen (I type, IV type) and gelatin as raw material, its weight is respectively 1150mg, 25mg, 575mg, and implementation process is with example 1;
Embodiment 3: adopt collagen (I type) and heparitin sulfate as raw material, its weight is respectively 1150mg, 0.5mg, and implementation process is with example 1;
Embodiment 4: adopt collagen (IV type) and heparitin sulfate as raw material, its weight be respectively 25mg, 0.5mg,, implementation process is with example 1;
Embodiment 5: adopt collagen (I type) and gelatin as raw material, its weight is respectively 1150mg, 575mg, and implementation process is with example 1;
Embodiment 6: adopt collagen (IV type) and gelatin as raw material, its weight is respectively 25mg, 575mg, and implementation process is with example 1;
Embodiment 7: adopt collagen (I type, IV type) and heparitin sulfate as raw material, its weight is respectively 1150mg, 25mg, 0.5mg, and implementation process is with example 1;
Embodiment 8: adopt collagen (IV type), gelatin and heparitin sulfate as raw material, its weight is respectively 25mg, 575mg, 0.5mg, and implementation process is with example 1.
The new type compound nervous tissue engineering repair materials that the present invention developed has the aperture and parallel, the single-orientated Y-direction micropore uniformly of the various sizes of internal diameter size between 20 μ m～300 μ m that can simple and easy regulation and control.This material also can be made different external forms easily, as column type, and oval column type etc.In the application of bioengineered tissue material, the attached wall of the pair cell of larger aperture is creeped or is poured into as Olfactory essheathing cell, schwann cell etc. and play support, carrier function; Smaller aperture due then be suitable for compound various nerve growth factor, medicine etc., as the carrier of its slow release, be used for the bridge joint at spinal cord or peripheral nervous tissue defect place.The material surface structure that the present invention developed approaches normal neural and does not have porous dehiscence, makes and implants spinal cord or peripheral nervous tissue defect place, can prevent that the hypertrophy connective tissue from growing into damaged section, the oriented growth of the regenerated fiber that affects the nerves.Be widely used in the experimentation of the clinical treatment of human peripheral nerve or segments of spinal cord damage and animal peripheral nervous or segments of spinal cord damage.
Claims (2)
1. a compound tissue engineering nerve injury repairing material is characterized in that it comprises: type i collagen albumen, heparitin sulfate, IV collagen type and gelatin; The percentage by weight of its prescription is:
Type i collagen albumen: 65.53%～65.71%;
Heparitin sulfate: 0.01%～0.28%;
IV collagen type: 1.42%～1.43%;
Gelatin: 32.77%～32.85%.
2. realize the described compound tissue engineering nerve injury repairing preparation methods of claim 1, under the thermograde condition, pass through to adjust the stranguria of cold type speed and the thermograde of raw mix, reach preparation inside and have the evenly distributed micro-tubular structure of axially parallel, and the microtubule diameter has special nerve injury, the impairment renovation material of controllability; It is characterized in that, carry out according to the following steps:
1) percentage by weight of formula for raw stock:
Type i collagen albumen: 65.53%
Heparitin sulfate: 0.28%
IV collagen type: 1.42%
Gelatin: 32.77%
2) take by weighing type i collagen albumen by above-mentioned ratio, put into the 0.05M acetum by the concentration of 27.5mg/ml and dissolve 24h;
3) in 4 ℃ of isoperibols, 18000r/min stirs 90min, makes suspension;
4) other takes by weighing in heparitin sulfate, the IV collagen type adding 0.05M acetum by above-mentioned ratio and dissolved 24 hours;
5) at 4 ℃ of constant temperature, 18000r/min stirs 90min and makes suspension;
6) mix two kinds of suspensions and keep 4 ℃ of constant temperature, 18000r/min stirs 90min, makes the suspension of collagen protein and heparitin sulfate, and evacuation leaves standstill 12h;
7) the bridge material suspension for preparing being injected internal diameter is that 3mm, external diameter are the silica gel tube sealing both ends of 4.2mm, long 3cm to 20cm, slowly puts into the agent of profound hypothermia stranguria of cold type along tube axial direction, and admission velocity is controlled to be 10 * 10
-5Ms
-1, 5 * 10
-5Ms
-1To 10 * 10
-7Ms
-1
8) suspension-silica gel tube frozen material is put into the good aluminum dish of pre-cooling, lyophilizing 48h in-30 ℃, 100mtorr condition;
9) be warming up to 0 ℃ under the vacuum state and keep 24h, continue to be warming up to 22 ℃ again and keep 45min～90min, remove vacuum, rise to room temperature;
10) material is soaked 24h with 20% glutaraldehyde and solidify, to the distilled water dialysis, again through the crosslinked 16h of ultraviolet radiation;
11) with the material aseptic sealed packages and place 2000 roentgen CO
60Illumination-based disinfection 24h in the environment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310118994 CN1256155C (en) | 2003-12-15 | 2003-12-15 | Tissue engineering compound material for repairing nerve injury and its preparation |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310118994 CN1256155C (en) | 2003-12-15 | 2003-12-15 | Tissue engineering compound material for repairing nerve injury and its preparation |
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|---|---|
| CN1546182A true CN1546182A (en) | 2004-11-17 |
| CN1256155C CN1256155C (en) | 2006-05-17 |
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|---|---|---|---|
| CN 200310118994 Expired - Fee Related CN1256155C (en) | 2003-12-15 | 2003-12-15 | Tissue engineering compound material for repairing nerve injury and its preparation |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1326576C (en) * | 2005-09-07 | 2007-07-18 | 中国科学院遗传与发育生物学研究所 | Biological restoration material, prepn. method and applicatino thereof |
| CN101653366B (en) * | 2009-06-11 | 2011-06-08 | 广州中大中山医科技开发有限公司 | Construction of gelatin sponge cylinder bracket used for repairing nerve injury |
| CN101766836B (en) * | 2009-01-21 | 2012-09-05 | 丁坦 | Preparation method of nano silver cordspinal cord and peripheral nerve repairing material |
| CN102671238A (en) * | 2009-01-16 | 2012-09-19 | 中国人民解放军第四军医大学 | High-emulation tissue-engineered nerve repair material |
| CN102727936A (en) * | 2012-06-20 | 2012-10-17 | 中山大学 | Construction method of sustained-release NT-3 gelatin sponge cylindrical stent used for repairing spinal cord injuries |
| CN102688523B (en) * | 2009-01-16 | 2014-04-16 | 中国人民解放军第四军医大学 | High-simulation tissue engineering nerve repair material |
| CN102671237B (en) * | 2009-01-16 | 2014-04-16 | 中国人民解放军第四军医大学 | High-simulation tissue engineering nerve-repair material and preparation method |
| CN103933619A (en) * | 2014-04-29 | 2014-07-23 | 深圳清华大学研究院 | Nerve repairing material and preparation method thereof |
| CN105327392A (en) * | 2014-08-08 | 2016-02-17 | 中国科学院遗传与发育生物学研究所 | Orderly collagen material, and preparation method and application thereof |
| CN115869466A (en) * | 2021-09-27 | 2023-03-31 | 中国科学院理化技术研究所 | Nerve repair tube, preparation and application thereof and mold for preparing nerve repair tube |
-
2003
- 2003-12-15 CN CN 200310118994 patent/CN1256155C/en not_active Expired - Fee Related
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1326576C (en) * | 2005-09-07 | 2007-07-18 | 中国科学院遗传与发育生物学研究所 | Biological restoration material, prepn. method and applicatino thereof |
| CN102671238A (en) * | 2009-01-16 | 2012-09-19 | 中国人民解放军第四军医大学 | High-emulation tissue-engineered nerve repair material |
| CN102688523B (en) * | 2009-01-16 | 2014-04-16 | 中国人民解放军第四军医大学 | High-simulation tissue engineering nerve repair material |
| CN102671237B (en) * | 2009-01-16 | 2014-04-16 | 中国人民解放军第四军医大学 | High-simulation tissue engineering nerve-repair material and preparation method |
| CN102671238B (en) * | 2009-01-16 | 2014-04-16 | 中国人民解放军第四军医大学 | High-emulation tissue-engineered nerve repair material |
| CN101766836B (en) * | 2009-01-21 | 2012-09-05 | 丁坦 | Preparation method of nano silver cordspinal cord and peripheral nerve repairing material |
| CN101653366B (en) * | 2009-06-11 | 2011-06-08 | 广州中大中山医科技开发有限公司 | Construction of gelatin sponge cylinder bracket used for repairing nerve injury |
| CN102727936A (en) * | 2012-06-20 | 2012-10-17 | 中山大学 | Construction method of sustained-release NT-3 gelatin sponge cylindrical stent used for repairing spinal cord injuries |
| CN103933619A (en) * | 2014-04-29 | 2014-07-23 | 深圳清华大学研究院 | Nerve repairing material and preparation method thereof |
| CN103933619B (en) * | 2014-04-29 | 2017-02-15 | 深圳清华大学研究院 | Nerve repairing material and preparation method thereof |
| CN105327392A (en) * | 2014-08-08 | 2016-02-17 | 中国科学院遗传与发育生物学研究所 | Orderly collagen material, and preparation method and application thereof |
| CN115869466A (en) * | 2021-09-27 | 2023-03-31 | 中国科学院理化技术研究所 | Nerve repair tube, preparation and application thereof and mold for preparing nerve repair tube |
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|---|---|
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