CN104606713A - Three-dimensional parallel collagenous fiber-silk bracket as well as preparation method and application thereof - Google Patents
Three-dimensional parallel collagenous fiber-silk bracket as well as preparation method and application thereof Download PDFInfo
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- CN104606713A CN104606713A CN201510005188.3A CN201510005188A CN104606713A CN 104606713 A CN104606713 A CN 104606713A CN 201510005188 A CN201510005188 A CN 201510005188A CN 104606713 A CN104606713 A CN 104606713A
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Abstract
The invention provides a three-dimensional parallel collagenous fiber-silk bracket as well as a preparation method thereof. The bracket is formed by a woven silk base frame and a parallel collagenous fiber layer which coats on the upper and lower surfaces of the woven silk base frame, wherein the woven silk base frame adopts a mesh structure which is woven by silk and has a mesh size of 0.25-25 mm<2>; the pore diameter of the parallel collagenous fiber layer is 10-300 microns; the woven silk is sericin-removed woven silk; the bracket can be used as a biological bracket and applied to the repair of muscle tendons/ligaments. The three-dimensional parallel collagenous fiber-silk bracket provided by the invention is simple to prepare, good in biocompatibility and good in mechanical property, has a topological structure similar to muscle tendons/ligaments and can be used for promoting the surface grown stem cells to differentiate toward the muscle tendons/ligaments and promoting the regeneration of the muscle tendons/ligaments.
Description
(1) technical field
The present invention relates to timbering material of a kind of three-dimensional for tendon/ligament repair and preparation method thereof, particularly three-dimensional parallel collagen fiber-silk stent of one and its preparation method and application.
(2) background technology
Along with the raising of China's expanding economy and living standards of the people, the concept of health of people is also in lifting, and for the yearning to healthy physique, the quantity that compatriots participate in physical training and competition obviously increases, athletic injury also gets more and more, and wherein ligament/tendon injury accounts for more than 50%.There are data to show, within every 200,000,000 populations 1 year, have up to ten million tendon injury cases at least.
Current, main directly stitching, autotransplantation, allograft, xenotransplantation and the prosthetic material prothesis of leaning on repairs ligament and tendon injury clinically.But, the defect that these operative reconstruction regeneration techniqueses have it intrinsic, such as recurrent is torn, donor shortage, donor site complication, immunologic rejection and poor support conformability.
The shortcoming of current clinical method impels research steering degradable biomaterial and the Method of Tissue Engineering of tendon/ligament repair.Use macromolecular material fiber or silkworm silk to form pigtail support for repairing damage tendon by researcher, but these pigtail supports not having internal voids, can not carry a large amount of cell, body can not be allowed to form the new organization being interconnected function.Researcher is had to adopt collagen to inoculate a large amount of cell for tendon/ligament tissue engineering in addition.These supports solve cell tissue and hold problem, but their mechanical property is very poor, can not bear the physiology mechanical force of body, are difficult to be applied to clinical.
There is research that silkworm silk and collagen sponge are combined with each other before, constructed a practical tendon/ligament tissue engineering rack.The space that collagen sponge provides repair cell to adhere to and grow into, braided silk fiber provides enough mechanical strengths.But it differs greatly with the shape of tendon/ligament tissue in structural arrangement, the 26S Proteasome Structure and Function repairing tendon/ligament, compared with normal tendon/ligament, also differs greatly.A kind of desirable, object cell can be attracted to gather at damage location and can effectively guide the combination of bioactive silk stent that has of cell migration to be still faced with huge challenge.
(3) summary of the invention
The object of the invention is to provide a kind of three-dimensional parallel collagen fiber-silk stent and preparation method thereof and application, support of the present invention has good mechanical property, the space that repair cell adheres to and grows into can be provided, there is the structure similar to tendon/ligament, can effectively guide cell migration and arrangement, can be used as the reparation that biological support is applied to tendon/ligament.
The technical solution used in the present invention is:
A kind of three-dimensional parallel collagen fiber-silk stent, described support is formed by weaving silkworm silk pedestal and being coated on the parallel collagen fiber layer weaving silkworm silk pedestal upper and lower surface, and described braiding silkworm silk pedestal is that to be woven into mesh size by silkworm silk be 0.25 ~ 25mm
2mesh sheet structure, the pore diameter size of described parallel collagen fiber is 10 ~ 300um; Described braiding silkworm silk is the braiding silkworm silk eliminating sericin.
The three-dimensional parallel collagen fiber-silk stent of the present invention, the mesh size of preferred described braiding silkworm silk pedestal is 1 ~ 9mm
2.
The pore diameter size of preferred described parallel collagen fiber is 50 ~ 150um.
Present invention also offers the preparation method of the parallel collagen fiber-silk stent of a kind of described three-dimensional, described preparation method comprises the steps:
(1) silkworm silk pedestal will be woven and remove sericin;
(2) eliminating above the braiding silkworm silk pedestal of sericin, below respectively implantation concentration be the collagen solution of 5 ~ 20mg/ml, and the thickness making braiding silkworm silk pedestal both sides collagen solution layer is 1 ~ 10mm;
(3) sub-cooled makes collagen set, and then vacuum decompression is dry;
(4) finally carry out dehydrothermal, obtain the parallel collagen fiber-silk stent of described three-dimensional.
In step of the present invention (1), the method that described braiding silkworm silk pedestal removes sericin can be selected from one of following: sodium carbonate liquor boils removal method, detergent boils removal method, boric acid boils removal method or direct heating removal method etc., these remove the method for sericin, are all applicable to the present invention.
Concrete, described sodium carbonate liquor boils removal method and is: braiding silkworm silk pedestal is immersed in 0.2wt%Na
2cO
3in aqueous solution, boil 60 ~ 150min, water is changed 2 ~ 5 times in centre, and room temperature or drying baker drying, must eliminate the braiding silkworm silk pedestal of sericin.
Concrete, described step (2) is carried out as follows: first, at one end being embedded with implantation concentration in the mould of temperature transfer bonding jumper is the collagen solution of 5 ~ 20mg/ml, and make sample path length be 1 ~ 10mm, keep flat the braiding silkworm silk pedestal eliminating sericin at solution upper surface after paving, then above braiding silkworm silk pedestal, implantation concentration is the collagen solution of 5 ~ 20mg/ml, and makes sample path length be 1 ~ 10mm, paves; Described temperature transfer bonding jumper is selected from aluminum strip, stainless steel strip or copper bar etc. and transmits the good material of temperature performance, and the material of described mould is selected from the material of the heat conductivility differences such as acrylic, pottery or silica gel.
In step of the present invention (3), the sub-cooled method of collagen set can be adopted one of following: the foam box of liquid nitrogen, refrigeration machine or low temperature ice cube etc. are housed, these manufacture thermogrades and make the method for collagen set, are all applicable to the present invention.
The three-dimensional parallel collagen fiber-silk stent of concrete recommendering folder invention is prepared as follows:
(1) braiding silkworm silk pedestal is immersed in 0.2wt%Na
2cO
3in aqueous solution, boil 60 ~ 150min, water is changed 2 ~ 5 times in centre, and room temperature or drying baker drying, obtain the braiding silkworm silk pedestal eliminating sericin;
(2) being at one end embedded with implantation concentration in the acrylic mould of aluminum strip is the collagen solution of 5 ~ 20mg/ml, and make sample path length be 1 ~ 10mm, keep flat at solution upper surface the braiding silkworm silk pedestal that step (1) gained eliminates sericin after paving, then above braiding silkworm silk pedestal, implantation concentration is the collagen solution of 5 ~ 20mg/ml, and make sample path length be 1 ~ 10mm, pave;
(3) be connected with the foam box that liquid nitrogen is housed by the other end of acrylic mould, adjustment foam box and the distance of mould are 10 ~ 100mm, after collagen all solidifies, are placed in vacuum and drain machine and drain;
(4) finally carry out dehydrothermal, method is: in the vacuum drying oven of <10mbar, places one day for 22 DEG C, places three days for 110 DEG C, places one day, obtains the parallel collagen fiber-silk stent of described three-dimensional for 65 DEG C.
The three-dimensional parallel collagen fiber-silk stent of the present invention can be used as the reparation that biological support is applied to tendon/ligament.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
1, the three-dimensional parallel collagen fiber-silk stent manufacture method of the present invention is simple, and its shape can control adjustment according to mould, and its hole can by temperature and collagen concentration adjustment;
2, the three-dimensional parallel collagen fiber-silk stent good biocompatibility of the present invention, collagen and silkworm silk all can be degraded in vivo;
3, the three-dimensional parallel collagen fiber-silk stent mechanical property of the present invention is good, can meet the powerful mechanics needed for tendon/ligament;
4, the topological structure of the three-dimensional parallel collagen fiber-silk stent of the present invention is similar to tendon/ligament, can promote that its epontic stem cell is to tendon/ligament differentiation, promotes tendon/ligament regeneration.
(4) accompanying drawing explanation
Fig. 1: braiding silkworm silk picture substantially;
Fig. 2: three-dimensional parallel collagen fiber-silk stent is schemed substantially;
Fig. 3: parallel collagen fiber electron-microscope scanning figure, Fig. 3-1 is 6mg/ml collagen, and Fig. 3-2 is 10mg/ml collagen, and Fig. 3-3 is 18mg/ml collagen;
Fig. 4: the propagation of tendon stem cell/precursor on the parallel collagen fiber-silk stent of three-dimensional;
Fig. 5: rabbit rotator cuff injury repairs HE dyeing after 4 weeks, and A is disorderly collagen fiber-silk stent group, and B is three-dimensional parallel collagen fiber-silk stent group;
Fig. 6: rabbit rotator cuff injury repairs Masson dyeing after 4 weeks, and A is disorderly collagen fiber-silk stent group, and B is three-dimensional parallel collagen fiber-silk stent group;
Fig. 7: rabbit rotator cuff injury repairs newborn tendon collagen content after 4 weeks;
Fig. 8: rabbit rotator cuff injury repairs tendon related gene expression after 4 weeks.
(5) detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1
Be 1mm by mesh size
2braiding silkworm silk remove sericin, be cut into 2*2cm (see Fig. 1).Below braiding silkworm silk, inject the collagen of the thick 6mg/ml of 2mm, above braiding silkworm silk, inject the collagen of the thick 6mg/ml of 3mm.The adjustment dress foam box of liquid nitrogen and the distance of mould are 5cm.Solidify and be placed on vacuum and drain in machine and drain, then carry out dehydrothermal, obtain the parallel collagen fiber-silk stent of three-dimensional that pore diameter size is about 200um.Observe its electron-microscope scanning picture (see Fig. 3-1).
Embodiment 2
Be 1mm by mesh size
2braiding silkworm silk remove sericin, be cut into 2*2cm.Below braiding silkworm silk, inject the collagen of the thick 10mg/ml of 2mm, above braiding silkworm silk, inject the collagen of the thick 10mg/ml of 3mm.The distance of the adjustment dress foam box of liquid nitrogen and collagen, silkworm silk is 5cm.Solidify and be placed on vacuum and drain in machine and drain, then carry out dehydrothermal, obtain the parallel collagen fiber-silk stent (see Fig. 2) of three-dimensional that pore diameter size is about 80um.Observe its electron-microscope scanning picture (see Fig. 3-2).
Embodiment 3
Be 1mm by mesh size
2braiding silkworm silk remove sericin, be cut into 2*2cm.Below braiding silkworm silk, inject the collagen of the thick 18mg/ml of 2mm, above braiding silkworm silk, inject the collagen of the thick 18mg/ml of 3mm.The distance of the adjustment dress foam box of liquid nitrogen and collagen, silkworm silk is 5cm.Solidify and be placed on vacuum and drain in machine and drain, then carry out dehydrothermal, obtain the parallel collagen fiber-silk stent of three-dimensional that pore diameter size is about 20um.Observe its electron-microscope scanning picture (see Fig. 3-3).
Embodiment 4
Tendon stem cell/precursor planted by the support of gained in example 2.Detected the proliferative conditions (see Fig. 4) of 1,3,7 day cell by cck-8 test kit, show cell normal proliferation on the parallel collagen fiber-silk stent of three-dimensional.
Embodiment 5
The application of three-dimensional parallel collagen fiber-silk stent in rotator cuff injury.
(1) set up animal model: by the female rabbits of 5 about 2500g, after intravenous anesthesia, cut skin, fascia and part muscle, supraspinatus tendon is fully exposed, press close to greater tubercles of humerus place cut-out supraspinatus tendon.
(2) stenter to implant: contrast about same rabbit, the support of gained in experimental group and embodiment 2, the collagen concentration of matched group and silkworm silk distribution, with embodiment 2, are directly put-20 DEG C of refrigerators and are solidified, lyophilizing, crosslinked with embodiment 2.By support one end directly and the supraspinatus tendon broken ends of fractured bone sew up, the other end is connected with greater tubercles of humerus by osseous tunnel.
(3) tissue repairing ability: postoperative 4 weeks, takes out rabbit supraspinatus tendon, carries out HE dyeing, Masson dyeing, Contents of collagen and tendon related gene and detects.
HE coloration result: experimental group internal stent also has newborn tendon to grow into, and the inner newborn tendon of crt bracket grows into seldom (see Fig. 5).Result shows that three-dimensional parallel collagen fiber-silk stent can promote that tendon regenerates.
Masson coloration result: the newborn tendon of experimental group internal stent is more than matched group, more ripe (see Fig. 6).Result shows that the facilitation of three-dimensional parallel collagen fiber-silk stent tendon regeneration is stronger than disorderly collagen fiber-silk stent.
Contents of collagen: the collagen content of experimental group is (131.12 ± 36.73) mg/g, matched group is (121.32 ± 27.12) mg/g (see Fig. 7), show that three-dimensional parallel collagen fiber-silk stent can promote more collagenation, promote tendon regeneration.
Tendon related gene expression: Col1, Col3, Tnc, Bgn all have higher expression (see Fig. 8) at experimental group.Show that three-dimensional parallel collagen fiber-silk stent can promote tendon related gene expression, thus promote tendon regeneration.
Claims (10)
1. the parallel collagen fiber-silk stent of three-dimensional, it is characterized in that, described support is formed with the parallel collagen fiber layer being coated on braiding silkworm silk pedestal upper and lower surface by weaving silkworm silk pedestal, and described braiding silkworm silk pedestal is that to be woven into mesh size by silkworm silk be 0.25 ~ 25mm
2mesh sheet structure, the pore diameter size of described parallel collagen fiber is 10 ~ 300um; Described braiding silkworm silk is the braiding silkworm silk eliminating sericin.
2. three-dimensional parallel collagen fiber-silk stent as claimed in claim 1, is characterized in that, the mesh size of described braiding silkworm silk pedestal is 1 ~ 9mm
2.
3. three-dimensional parallel collagen fiber-silk stent as claimed in claim 1, is characterized in that, the pore diameter size of described parallel collagen fiber is 50 ~ 150um.
4. a preparation method for the parallel collagen fiber-silk stent of the three-dimensional as described in one of claims 1 to 3, it is characterized in that, described preparation method comprises the steps:
(1) silkworm silk pedestal will be woven and remove sericin;
(2) eliminating above the braiding silkworm silk pedestal of sericin, below respectively implantation concentration be the collagen solution of 5 ~ 20mg/ml, and the thickness making braiding silkworm silk pedestal both sides collagen solution layer is 1 ~ 10mm;
(3) sub-cooled makes collagen set, and then vacuum decompression is dry;
(4) finally carry out dehydrothermal, obtain the parallel collagen fiber-silk stent of described three-dimensional.
5. preparation method as claimed in claim 4, it is characterized in that in step (1), the method that described braiding silkworm silk pedestal removes sericin is selected from one of following: sodium carbonate liquor boils removal method, detergent boils removal method, boric acid boils removal method or direct heating removal method.
6. preparation method as claimed in claim 5, it is characterized in that, described sodium carbonate liquor boils removal method and is: braiding silkworm silk pedestal is immersed in 0.2wt%Na
2cO
3in aqueous solution, boil 60 ~ 150min, water is changed 2 ~ 5 times in centre, and room temperature or drying baker drying, must eliminate the braiding silkworm silk pedestal of sericin.
7. preparation method as claimed in claim 4, it is characterized in that, described step (2) is carried out as follows: first, at one end being embedded with implantation concentration in the mould of temperature transfer bonding jumper is the collagen solution of 5 ~ 20mg/ml, and make sample path length be 1 ~ 10mm, keep flat the braiding silkworm silk pedestal eliminating sericin at solution upper surface after paving, then above braiding silkworm silk pedestal, implantation concentration is the collagen solution of 5 ~ 20mg/ml, and make sample path length be 1 ~ 10mm, pave; Described temperature transfer bonding jumper is selected from aluminum strip, stainless steel strip or copper bar, and the material of described mould is selected from acrylic, pottery or silica gel.
8. preparation method as claimed in claim 4, is characterized in that in described step (3), one of makes the employing of the sub-cooled method of collagen set following: the foam box of liquid nitrogen, refrigeration machine or low temperature ice cube are housed.
9. preparation method as claimed in claim 4, it is characterized in that, described preparation method is carried out as follows:
(1) braiding silkworm silk pedestal is immersed in 0.2wt%Na
2cO
3in aqueous solution, boil 60 ~ 150min, water is changed 2 ~ 5 times in centre, and room temperature or drying baker drying, obtain the braiding silkworm silk pedestal eliminating sericin;
(2) being at one end embedded with implantation concentration in the acrylic mould of aluminum strip is the collagen solution of 5 ~ 20mg/ml, and make sample path length be 1 ~ 10mm, keep flat at solution upper surface the braiding silkworm silk pedestal that step (1) gained eliminates sericin after paving, then above braiding silkworm silk pedestal, implantation concentration is the collagen solution of 5 ~ 20mg/ml, and make sample path length be 1 ~ 10mm, pave;
(3) be connected with the foam box that liquid nitrogen is housed by the other end of acrylic mould, adjustment foam box and the distance of mould are 10 ~ 100mm, after collagen all solidifies, are placed in vacuum and drain machine and drain;
(4) finally carry out dehydrothermal, method is: in the vacuum drying oven of <10mbar, places one day for 22 DEG C, places three days for 110 DEG C, places one day, obtains the parallel collagen fiber-silk stent of described three-dimensional for 65 DEG C.
10. the parallel collagen fiber-silk stent of the three-dimensional as described in one of claims 1 to 3 is applied to the reparation of tendon/ligament as biological support.
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Cited By (8)
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| CN107469147A (en) * | 2017-08-14 | 2017-12-15 | 浙江大学 | A kind of silk collagen scaffold of biotic factor release and preparation method and application |
| CN108653811A (en) * | 2017-03-28 | 2018-10-16 | 上海微创医疗器械（集团）有限公司 | A kind of artificial ligament and preparation method thereof |
| CN109758262A (en) * | 2018-12-29 | 2019-05-17 | 浙江星月生物科技股份有限公司 | The adjustable braided silk stent of mechanics degradation property and preparation method and application |
| CN110947032A (en) * | 2019-12-16 | 2020-04-03 | 浙江大学 | Silk/PET mixed-woven bracket and preparation method and application thereof |
| CN113559329A (en) * | 2021-09-03 | 2021-10-29 | 北京大学口腔医学院 | Periodontal-imitated ordered double-layer structure stent material and preparation method and application thereof |
| CN113944003A (en) * | 2020-10-28 | 2022-01-18 | 清华大学 | Multi-scale tissue engineering composite scaffold and preparation device and preparation method thereof |
| CN115315277A (en) * | 2020-01-31 | 2022-11-08 | 恩博迪股份有限公司 | Braided surgical implant |
| CN115845139A (en) * | 2022-12-08 | 2023-03-28 | 海南宏正生物科技有限公司 | Artificial skin for promoting tissue repair and regeneration and preparation method thereof |
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Cited By (11)
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| CN108653811A (en) * | 2017-03-28 | 2018-10-16 | 上海微创医疗器械（集团）有限公司 | A kind of artificial ligament and preparation method thereof |
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| CN109758262A (en) * | 2018-12-29 | 2019-05-17 | 浙江星月生物科技股份有限公司 | The adjustable braided silk stent of mechanics degradation property and preparation method and application |
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| CN115315277A (en) * | 2020-01-31 | 2022-11-08 | 恩博迪股份有限公司 | Braided surgical implant |
| CN113944003A (en) * | 2020-10-28 | 2022-01-18 | 清华大学 | Multi-scale tissue engineering composite scaffold and preparation device and preparation method thereof |
| CN113944003B (en) * | 2020-10-28 | 2024-01-26 | 清华大学 | Multi-scale tissue engineering composite scaffold and preparation device and preparation method thereof |
| CN113559329A (en) * | 2021-09-03 | 2021-10-29 | 北京大学口腔医学院 | Periodontal-imitated ordered double-layer structure stent material and preparation method and application thereof |
| CN115845139A (en) * | 2022-12-08 | 2023-03-28 | 海南宏正生物科技有限公司 | Artificial skin for promoting tissue repair and regeneration and preparation method thereof |
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