CN105903071A - Cornea scaffold material and preparation method thereof and 3D printing method of cornea scaffold - Google Patents
Cornea scaffold material and preparation method thereof and 3D printing method of cornea scaffold Download PDFInfo
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Abstract
The invention provides a cornea scaffold material and its preparation method and a 3D printing method of a cornea scaffold. A technical effect of preparing a cornea scaffold by a 3D printing technology is achieved. Silkworm cocoon undergoes degumming by the use of a sodium carbonate aqueous solution; the silkworm cocoon is dissolved with a lithium bromide aqueous solution, and dialysis, suction filtration and centrifugal treatment are successively carried out to obtain a concentrated silk fibroin aqueous solution, a silk fibroin nano-structure is formed and extracellular matrix structure can be simulated; a graphene oxide aqueous solution which has undergone ultrasonic dispersion treatment is mixed with the silk fibroin aqueous solution, and mass ratio of silk fibroin to graphene oxide is 10000:1-1000:1 to raise mechanical strength and transparency of the material; and a gelatin aqueous solution and a cross-linking agent are added into the obtained mixed aqueous solution, and the mixed aqueous solution is uniformly stirred at 30-50 DGE C to regulate degradation speed of the material and guarantee that the cornea scaffold can be printed. Therefore, the cornea scaffold can be printed by the 3D printing technology.
Description
Technical field
The invention belongs to embedded material and organ technical field in medical apparatus and instruments, specifically, relate to the 3D Method of printing of a kind of cornea scaffold and preparation method thereof and CF.
Background technology
The corneal stroma of the mankind accounts for the 90% of corneal thickness, mainly formed by the extracellular matrix components such as keratocyte and collagen fiber is regularly arranged, transparent and there is certain radius of curvature, be conducive to light to pass through and warpage, at aspects such as maintaining the transparency of whole cornea and bent light rate, there is vital effect.
Most corneal stromas sick blind patient all can be cured by corneal graft, but cannot recover lost eyesight owing to the height scarcity of donation cornea causes Most patients to can not get donor's cornea.
In recent years, three-dimensional printing technology is developed rapidly in terms of preparing used in tissue engineering three-dimensional rack and applies, by controlling print platform cryogenic temperature, make material molecule freeze forming within a short period of time, natural biologic material especially for some easy degeneration, the natural biologic material liquid quick freezing molding of not easy-formation can be made and its conformation in the solution and biological activity can be retained, this technology makes the reconstruction in vitro of the structure corneal stroma stent similar with natural cornea structure and clinical practice thereof be possibly realized, also see light again for the sick blind patient of corneal stroma and create condition.
Summary of the invention
The invention provides the 3D Method of printing of a kind of cornea scaffold and preparation method thereof and CF, it is achieved be to use 3D printing technique to prepare the technique effect of CF.
For solving above-mentioned technical problem, the present invention is achieved by the following technical solutions:
A kind of cornea scaffold is proposed, the following raw material including dividing by quality: the aqueous gelatin solution of 30%-70%, fibroin albumen and graphene oxide mixed aqueous solution and the cross-linking agent of 0.1%-0.5% of 29.5%-69.9%；Wherein, described fibroin albumen is with graphene oxide mixed aqueous solution, and fibroin albumen is 10000:1-1000:1 with the mass ratio of graphene oxide.
Further, in institute's gelatin water solution, the quality accounting of gelatin is 30%-60%.
Proposing the preparation method of a kind of cornea scaffold, described cornea scaffold includes the following raw material divided by quality: the aqueous gelatin solution of 30%-70%, fibroin albumen and graphene oxide mixed aqueous solution and the cross-linking agent of 0.1%-0.5% of 29.5%-69.9%；Wherein, described fibroin albumen is with graphene oxide mixed aqueous solution, and silk fibroin water solution is 10000:1-1000:1 with the mass ratio of graphene oxide water solution；Comprise the steps: 1) with aqueous sodium carbonate to degumming silkworm cocoons after, dissolved with lithium bromide water solution, then obtained silk fibroin water solution through dialysis, sucking filtration and centrifugal treating；2), after graphene oxide water solution being carried out ultrasonic disperse process, it is mixed to get mixed aqueous solution with the silk fibroin water solution that obtains in step 1)；In described mixed aqueous solution, fibroin albumen is 10000:1-1000:1 with the mass ratio of graphene oxide；3) in step 2) in the mixed aqueous solution that obtains adds aqueous gelatin solution and cross-linking agent, and stir under conditions of temperature is 30 DEG C-50 DEG C；Wherein, institute's gelatin water solution quality accounting in blend solution is 30%-70%, and described cross-linking agent quality accounting in blend solution is 0.1%-0.5%.
Further, the dialysis treatment in step 1) includes: dialysed in deionized water 72 hours by the bag filter that lithium bromide water solution molecular cut off is 3500 being dissolved with degumming Bombyx bombycis;Period, within every 2 hours, change water once.
Further, in step 1), after dialysis, sucking filtration and centrifugal treating, need to concentrate in the environment of 10 DEG C-78 DEG C and within 3-200 hour, obtain described silk fibroin water solution.
Further, in step 3), in institute's gelatin water solution, the quality accounting of gelatin is 30%-60%.
Propose the 3D printing preparation method of a kind of CF, comprise the steps: in the shower nozzle of biometric print machine, load the cornea scaffold prepared according to the preparation method of above-mentioned cornea scaffold；Under conditions of 50 DEG C, successively print CF according to setting program.
Compared with prior art, advantages of the present invention and good effect be: in the 3D Method of printing of cornea scaffold that the present invention proposes and preparation method thereof and CF, fibroin albumen and the mixed aqueous solution of graphene oxide, inherently make use of the self-assembly property of fibroin albumen, form it into nanostructured, imitate the structure of extracellular matrix, improve the biocompatibility of cornea scaffold；Graphene oxide can change the secondary structure of fibroin albumen and promote the formation of fibroin albumen crystalline texture, it is possible to increase the mechanical strength of cornea scaffold and the transparency；The mixing of aqueous gelatin solution and the regulation and control of ratio, improve the problem of gelatin degradation excessive velocities simultaneously, improve coercibility and the toughness of cornea scaffold, ensure that cornea scaffold can printing shaping, make this cornea scaffold can be applied to 3D printing technique, realized the preparation of CF by 3D printing technique.
Using the cornea scaffold that the present invention proposes, by the CF of 3D printing technique printing shaping, biological degradability performance good, bionical is excellent, mechanical strength is high and transparent good, can preferably promote the reparation of cornea after using.
After reading in conjunction with the accompanying the detailed description of embodiment of the present invention, the other features and advantages of the invention will become clearer from.
Accompanying drawing explanation
Fig. 1 is the flow chart of the 3D Method of printing of the preparation method of the cornea scaffold that the present invention proposes and CF；
Fig. 2 is the scanning electron microscope (SEM) photograph of the cornea scaffold according to embodiment one preparation；
Fig. 3 is the scanning electron microscope (SEM) photograph of the cornea scaffold according to embodiment three preparation.
Detailed description of the invention
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described in more detail.
The present invention proposes a kind of cornea scaffold, the following raw material including dividing by quality: the aqueous gelatin solution of 30%-70%, fibroin albumen and graphene oxide mixed aqueous solution and the cross-linking agent of 0.1%-0.5% of 29.5%-69.9%；Wherein, fibroin albumen is with graphene oxide mixed aqueous solution, and fibroin albumen is 10000:1-1000:1 with the mass ratio of graphene oxide.Wherein, in aqueous gelatin solution, the quality accounting of gelatin is 30%-60%.
The preparation method of this cornea scaffold, as it is shown in figure 1, comprise the steps:
Step S1: with aqueous sodium carbonate to degumming silkworm cocoons after, dissolved with lithium bromide water solution, then obtained silk fibroin water solution through dialysis, sucking filtration and centrifugal treating.
Here, the mass percent of calcium carbonate aqueous solution is 0.5%-1%, and the molar concentration of lithium bromide water solution is 9.5 ± 0.3mol/L.
Dialysis treatment is particularly as follows: dialyse the bag filter that lithium bromide water solution molecular cut off is 3500 being dissolved with degumming Bombyx bombycis in deionized water 72 hours;Period, within every 2 hours, change water once.
At the silk fibroin water solution obtained after dialysis, sucking filtration and centrifugal treating, concentrate 3-200 hour in the environment of also needing to be placed on 10 DEG C-78 DEG C, obtain is the silk fibroin water solution concentrated, and in the silk fibroin water solution of this concentration, the quality accounting of fibroin albumen is 10%-30%.By the regulation and control to fibroin albumen so that it is form nanostructured, it is possible to make silk fibroin protein solution carry out self assembly, thus obtain class ECM(extracellular matrix) structure, improves biocompatibility.
The silk fibroin water solution obtained in step S2: after graphene oxide water solution is carried out ultrasonic disperse process, with step S1 is mixed to get mixed aqueous solution, and in this mixed aqueous solution, fibroin albumen is 10000:1-1000:1 with the mass ratio of graphene oxide.
The concentration of graphene oxide water solution is 0.1-5mg/ml, and the ultrasonic time that graphene oxide water solution carries out ultrasonic disperse process controlled as 2-20 minute.
The mixed aqueous solution obtained in this step, substantially can change the secondary structure of fibroin albumen, promotes the formation of fibroin albumen crystalline texture, can not only improve mechanical strength and the stability of fibroin material, it is ensured that material can printing shaping；Simultaneously as the high index of refraction characteristic of graphene oxide so that the cornea scaffold transparency of acquisition improves.
Step S3: add aqueous gelatin solution and cross-linking agent in the mixed aqueous solution obtained in step s 2, and stir under conditions of temperature is 30 DEG C-50 DEG C.
Among these, aqueous gelatin solution quality accounting in blend solution is 30%-70%, and cross-linking agent quality accounting in blend solution is 0.1%-0.5%.And the quality accounting of gelatin is 30%-60% in aqueous gelatin solution.
During gelatin is blended with fibroin albumen and graphene oxide, the degradation speed being regulated and controled cornea scaffold by the ratio controlling fibroin albumen and gelatin meets the needs that artificial cornea repairs.Meanwhile, gelatin improves coercibility and the toughness of cornea scaffold, it is ensured that cornea scaffold can printing shaping.
After ensure that the mechanical property of cornea scaffold, transparency, degradation speed, coercibility and toughness, this cornea scaffold can be applied to 3D printing technique, prints CF by 3D printing technique.
Based on above-mentioned, the present invention also proposes the 3D Method of printing of a kind of CF, as it is shown in figure 1, comprise the steps:
Step S4: load cornea scaffold in the shower nozzle of biometric print machine.
This cornea scaffold is the above-mentioned cornea scaffold prepared according to the preparation method of the cornea scaffold of step S1-step S31.
Step S5: under conditions of 50 DEG C, successively prints CF according to setting program.
Concrete printing, according to print parameters demand, realizes with the print procedure of existing biometric print machine, and the embodiment of the present invention not limits.
Below with some specific embodiments, the 3D printing preparation method of cornea scaffold that the present invention proposes and preparation method thereof and CF is illustrated.
Implement one
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.
Above-mentioned silk fibroin protein solution carries out in the environment of 60 DEG C concentration, and concentration time is 120 hours, and after fibroin albumen concentrates, solution concentration is 10%～about 30%.
It is that 2mg/ml graphene oxide water solution is made to mix with silk fibroin water solution after ultrasonic disperse processes by concentration, shakes up；Wherein, graphene oxide is 1:10000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 1:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Above-mentioned blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 75MPa, and elongation at break is 50%, and energy to failure is 8J/g.
As shown in Figure 2, in the present embodiment one, fibroin albumen after concentrated process is blended with graphene oxide and aqueous gelatin solution and prints biological response of synthetic materials used for keratoprosthesis surface topography map, there is a lot of fibrous structure on its surface visible, and the structure of this kind ECM can promote cell growth on material.
Embodiment two
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.
Above-mentioned silk fibroin protein solution carries out in the environment of 60 DEG C concentration, and concentration time is 120 hours, and after fibroin albumen concentrates, solution concentration is 10%～about 30%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:1000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 1:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 80MPa, and elongation at break is 70%, and energy to failure is 10J/g.
Embodiment three
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:10000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 1:1 add aqueous gelatin solution forms red miscible fluid, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 73MPa, and elongation at break is 62%, and energy to failure is 13J/g.
As it is shown on figure 3, be that the fresh silk fibroin solution described in embodiment three is blended with graphene oxide and gelatin solution print biological response of synthetic materials used for keratoprosthesis surface topography map, its surface does not have fibrous structure.
Embodiment four
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:1000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 1:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 68MPa, and elongation at break is 67%, and energy to failure is 15J/g.
Embodiment five
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.Above-mentioned silk fibroin protein solution carries out in the environment of 60 DEG C concentration, and concentration time is 120 hours, and after fibroin albumen concentrates, solution concentration is 10%～about 30%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:10000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 2:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 85MPa, and elongation at break is 73%, and energy to failure is 13J/g.
Embodiment six
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.Above-mentioned silk fibroin protein solution carries out in the environment of 60 DEG C concentration, and concentration time is 120 hours, and after fibroin albumen concentrates, solution concentration is 10%～about 30%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:1000's with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 2:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 83MPa, and elongation at break is 62%, and energy to failure is 20J/g.
Embodiment seven
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Take the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.Above-mentioned silk fibroin protein solution carries out in the environment of 60 DEG C concentration, and concentration time is 120 hours, and after fibroin albumen concentrates, solution concentration is 10%～about 30%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:10000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 2:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 86MPa, and elongation at break is 73%, and energy to failure is 20/g.
Embodiment eight
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in the LiBr solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.Above-mentioned silk fibroin protein solution carries out in the environment of 60 DEG C concentration, and concentration time is 120 hours, and after fibroin albumen concentrates, solution concentration is 10%～about 30%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:1000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 2:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 88MPa, and elongation at break is 69%, and energy to failure is 17J/g.
Embodiment nine
50g silkworm silk is put into the Na of 0.5%2CO3Solution carry out degumming process and dries, boiling 1 hour with 100 DEG C during degumming, to remove the sericin outside silkworm silk, using deionized water rinsing, will dry at silkworm silk 60 DEG C after more than repetition operating 3 times.
Weigh the boiled silk 15g after above-mentioned process to put into and carry out dissolution process in lithium bromide (LiBr) solution that 100mL concentration is 9.3mol/L, dissolve 4 hours at 60 DEG C during dissolution process.Then the above-mentioned LiBr solution being dissolved with silkworm silk is carried out dialysis treatment, soaking dialysis 3 days in deionized water with the bag filter of molecular cut off 3500 during dialysis, period every two hours changes a water, to remove the LiBr in solution, thus obtaining pure silk fibroin protein solution, its concentration is 6%.Above-mentioned silk fibroin protein solution carries out in the environment of 60 DEG C concentration, and concentration time is 120 hours, and after fibroin albumen concentrates, solution concentration is 10%～about 30%.
By concentration be 2mg/ml graphene oxide water solution make ultrasonic disperse process mix with silk fibroin water solution, shake up；Wherein, graphene oxide is 1:1000 with the mass ratio of fibroin albumen.Then according to fibroin albumen and gelatin mass ratio 2:1 add aqueous gelatin solution forms blend solution, and by the water-soluble carbodiimide of EDC(that mass ratio is 5:1): NHS(butanimide) add blend solution, make mixed solution cross-link.Finally, mix homogeneously under the conditions of 30～50 DEG C.
Blend solution is loaded in the shower nozzle of biometric print machine, under conditions of 50 DEG C, according to the program set, successively print, form required artificial cornea's material.By material 25 DEG C, relative humidity 50%, deposit balance under the conditions of constant temperature and humidity 48 hours, i.e. obtain described a kind of activeness and quietness regenerated silk fiber, its fracture strength is 83MPa, and elongation at break is 20%, and energy to failure is 3J/g.
It should be noted that; described above is not limitation of the present invention; the present invention is also not limited to the example above, change that those skilled in the art are made in the essential scope of the present invention, retrofits, adds or replaces, and also should belong to protection scope of the present invention.
Claims (7)
1. a cornea scaffold, it is characterised in that include the following raw material divided by quality: the aqueous gelatin solution of 30%-70%, fibroin albumen and graphene oxide mixed aqueous solution and the cross-linking agent of 0.1%-0.5% of 29.5%-69.9%；Wherein, described fibroin albumen is with graphene oxide mixed aqueous solution, and fibroin albumen water is 10000:1-1000:1 with the mass ratio of graphene oxide.
Cornea scaffold the most according to claim 1, it is characterised in that in institute's gelatin water solution, the quality accounting of gelatin is 30%-60%.
3. a preparation method for cornea scaffold, is used for preparing cornea scaffold as claimed in claim 1, it is characterised in that comprise the steps:
1) with aqueous sodium carbonate to degumming silkworm cocoons after, dissolved with lithium bromide water solution, then obtained silk fibroin water solution through dialysis, sucking filtration and centrifugal treating；
2), after graphene oxide water solution being carried out ultrasonic disperse process, it is mixed to get mixed aqueous solution with the silk fibroin water solution that obtains in step 1)；In described mixed aqueous solution, fibroin albumen is 10000:1-1000:1 with the mass ratio of graphene oxide；
3) in step 2) in the mixed aqueous solution that obtains adds aqueous gelatin solution and cross-linking agent, and stir under conditions of temperature is 30 DEG C-50 DEG C；Wherein, institute's gelatin water solution quality accounting in blend solution is 30%-70%, and described cross-linking agent quality accounting in blend solution is 0.1%-0.5%.
The preparation method of cornea scaffold the most according to claim 3, it is characterised in that the dialysis treatment in step 1) includes: the bag filter that lithium bromide water solution molecular cut off is 3500 being dissolved with degumming Bombyx bombycis is dialysed 72 hours in deionized water;Period, within every 2 hours, change water once.
The preparation method of cornea scaffold the most according to claim 3, it is characterised in that in step 1), after dialysis, sucking filtration and centrifugal treating, need to concentrate in the environment of 10 DEG C-78 DEG C and within 3-200 hour, obtain described silk fibroin water solution.
The preparation method of cornea scaffold the most according to claim 3, it is characterised in that in step 3), in institute's gelatin water solution, the quality accounting of gelatin is 30%-60%.
7. the 3D Method of printing of CF, it is characterised in that comprise the steps:
The cornea scaffold prepared according to the preparation method of the cornea scaffold described in any one of claim 3-6 is loaded in the shower nozzle of biometric print machine；
Under conditions of 50 DEG C, successively print CF according to setting program.
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| CN113750292A (en) * | 2021-09-30 | 2021-12-07 | 华南理工大学 | Biological ink for 3D printing of cornea repair material, preparation method of biological ink and preparation method of cornea repair material |
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