CN110665061A - Acellular scaffold solution-GelMA hydrogel composite material and preparation method thereof - Google Patents

Abstract

The invention discloses a decellularized scaffold solution-GelMA hydrogel composite material and a preparation method. The decellularized scaffold solution is mixed and dissolved in the GelMA hydrogel, and a porous structure is constructed by a combination of biochemical enzymatic hydrolysis and physical stirring. Composite stent material. The invention uses GelMA as the matrix to ensure the mechanical strength and biodegradability of the scaffold; the composite decellularized scaffold solution aims to improve the biocompatibility, retain the biological active factors and natural extracellular matrix components in the original organ, and promote the cells on the scaffold. Adhesion and proliferation. The composite scaffold prepared by the method has the advantages of adjustable pore size, good mechanical properties, good biocompatibility, and many active factors, and can be used as an excellent biological material in the field of tissue engineering.

Description

A kind of decellularized scaffold solution-GelMA hydrogel composite material and preparation method

technical field

The invention relates to the technical field of biological materials, in particular to a decellularized scaffold solution-GelMA hydrogel composite material and a preparation method.

Background technique

Extracellular matrix material is one of the main components of engineered tissue construction. The ideal scaffold material should meet the following characteristics: 1) It must have a three-dimensional porous network structure, which is conducive to cell growth and the transport of nutrients and metabolites, and the pore size and porosity are easy to control, creating conditions for angiogenesis; 2) It must have Degradability and good biocompatibility, scaffold space structure and surface chemical properties need to be adapted to cell adhesion, proliferation and differentiation; 3) Easy to process into various shapes and sizes; 4) Scaffolds of whole organs (envelopes and piping systems) ) organic integration with the extracellular matrix.

Recent studies have shown that as an extracellular matrix material, hydrogel can provide a suitable growth environment for the growth and differentiation of hepatocytes, and can support the long-term growth of hepatocytes in the scaffold. In addition, hydrogel materials all have fluidity and semi-solid state, which can not only be uniformly mixed with seed cells, but also can be implanted into the body by injection, thereby greatly reducing the damage to the pipeline and microenvironment of the implantation site.

Compared with synthetic hydrogels, natural hydrogels contain multiple integrated anchoring sites and growth factors, thus stimulating multiple signaling pathways including proliferation and differentiation. Among the natural hydrogels that can be used for liver tissue engineering are Matrigel, fibrin glue, collagen, chitosan, etc. In recent years, a new type of photosensitive hydrogel material, gelatin methacrylate anhydride (GelMA), has been widely favored by scholars. Its advantages include: the structure has cell adhesion sites and matrix metalloproteinase hydrolysis sites , so it can well support the proliferation and migration of cells; the existence of methacrylic anhydride group makes it photosensitive and can be quickly cross-linked under ultraviolet light; the physical and chemical properties are flexible and adjustable; Printing, lithography, self-assembly, microfluidics, etc., produce structural units with unique morphological characteristics. GelMA hydrogels have been proven to have unique advantages in tissue regeneration such as bone, cartilage, myocardium, and blood vessels, and have also achieved good results in basic cell research, cell signal transduction, drug/gene controlled release, and biosensing. result. However, these are single components, which cannot fully simulate the in vivo environment of organs, and have limited biological properties.

Therefore, using acellular technology to directly remove the cells and DNA that can cause immune rejection in the tissue, and use the remaining acellular matrix that retains most of the components of the natural tissue as a scaffold material, in various aspects of tissue engineering, such as nerve, liver , bone, muscle, muscle bond, heart and other aspects of the research is gradually increasing. Acellular matrices have already been marketed in applications such as skin regeneration/repair, nerve repair, anti-adhesion membranes, and tissue filling. Although these acellular matrix materials retain the main active ingredients, deformation and internal space collapse will inevitably occur during the decellularization process and after implantation. There are batch differences and it is difficult to achieve the same match.

Therefore, the present invention provides a rapid decellularization technology, and uses physical and chemical means to obtain acellular matrix solution and GelMA hydrogel to combine into a composite material, which retains the advantages of traditional hydrogel and increases the natural components of the decellularized material, It provides a new idea for the development of tissue engineering field.

SUMMARY OF THE INVENTION

The invention overcomes the shortcomings of single component and single biological function of traditional natural polymer gel. The traditional acellular matrix material has the disadvantages of easy deformation, large batch variation, poor processability, and difficult to fully match with the patient's wound. The present invention provides a preparation method of an acellular scaffold solution-GelMA hydrogel composite material. The preparation method of the invention is that the acellular matrix is powdered, digested and mixed with GelMA hydrogel to form a gel. The gel-forming technology can be used for both injectable gels and processable gels, and the gels formed have a porous structure, which has a positive effect on regulating cell behavior and making it fully contact with external nutrients.

For this reason, the present invention adopts the following technical solutions:

First, the present invention provides an acellular scaffold solution-GelMA hydrogel composite material, which is characterized by comprising the following components: acellular matrix, GelMA, and a cross-linking agent, wherein the acellular matrix exists in the acellular matrix liquid , the concentration of the acellular matrix in the acellular matrix solution is 1-2 mg/ml, GelMA exists in the 10% wt GelMA hydrogel prepared in distilled water, and the cross-linking agent is 1% wt photoinitiator, among which, GelMA hydrogel The mass ratio of gel to photoinitiator was 100:1, and the volume ratio of acellular matrix fluid to GelMA hydrogel was 1:1-1:9.

Meanwhile, the present invention also provides a preparation method of the decellularized scaffold solution-GelMA hydrogel composite material, the method comprising the following steps:

(1) Take the animal organs detached from the carrier and perfuse SDS and Triton X-100 aqueous solution at room temperature to remove the cells and nucleic acid substances in the tissue to obtain the decellularized scaffold;

(2) Freeze-dry the acellular scaffold obtained in step (1), cut into pieces to obtain acellular matrix block, and digest the matrix block with pepsin solution for 12-48 hours;

(3) Terminate the digestion with 10 x PBS, and add NaOH solution to adjust the pH of the decellularized matrix to neutral;

(4) Ultracentrifuge the neutral acellular matrix fluid with an ultrafiltration tube to obtain a concentrated decellularized matrix fluid;

(5) Mix the concentrated decellularized matrix solution and 10% by mass GelMA in a certain proportion, stir ultrasonically until uniform distribution, add the corresponding photoinitiator, and perform photocrosslinking under ultraviolet light to obtain the decellularized scaffold solution- GelMA hydrogel composites.

Further, the preparation method of the decellularized scaffold in the step (1) includes the following steps:

(1) Obtaining fresh organs: Take fresh and intact animal organs, cannulate them through veins or arteries, fix the cannula and vessels with hemostatic forceps, carefully take out the intact organs, place them on a sterile plate and rinse excess blood cells with PBS. Place in -80°C refrigerator, freeze and thaw repeatedly 2 to 5 times;

(2) Decellularization of organs: firstly perfuse PBS for 1~2h to remove blood cells, then perfuse 0.1% SDS for 6~10h, Triton X-100 for 0.5~1h, and lavage with a mixed solution of DNase and RNase The organs were removed for 10-30 min to remove excess nucleoplasm, and finally rinsed with PBS for 2-4 h, and stored in a mixed solution containing dual antibodies and amphotericin.

Further, in the step (1), the step of preparing the acellular matrix solution includes:

(1) Freeze-dry the decellularized scaffold, cut it into powder, digest the cut matrix with a hydrochloric acid solution of pepsin, and treat it on a shaker for 12 to 48 hours, until it dissolves into a homogeneous solution;

(2) Add 1/10~1/20 volume of 10 x PBS solution to the acellular matrix solution to stop the enzymatic digestion, and then add NaOH (5M) to make the acellular matrix solution neutral (pH=7.0~7.4) ;

(3) Transfer the neutralized acellular matrix solution to an ultrafiltration tube, centrifuge at 5000~6000 g at 4°C for 1~2 h, and collect the concentrated acellular matrix solution.

Further, the decellularization flow rate of the step (1) is 10 mL/min~20 mL/min.

Further, in the step (2), the pepsin solution is a hydrochloric acid solution containing pepsin, wherein the concentration of the hydrochloric acid solution is 0.1-0.5 M, and the concentration of pepsin is 0.5-5 mg/mL.

Further, the ratio of the amount of pepsin to the acellular matrix is 1-5 mg of the acellular scaffold dissolved in 1-10 mL of a pepsin hydrochloric acid solution.

Further, the concentration of the photoinitiator in the step (5) is 1-2% wt.

Further, in the step (5), the wavelength of the ultraviolet light is 365 nm, and the illumination time is 30 to 60 seconds.

Further, the mixing ratio of the acellular matrix solution in the step 5 and GelMA is greater than 1:1, wherein, the GelMA preparation method is that 0.1 g of GelMA is dissolved in 1 mL of an aqueous solution or a culture medium solution.

The decellularized scaffold solution-GelMA hydrogel composite material prepared by the invention has adjustable pores and is beneficial to the exchange of nutrients.

The above-mentioned decellularized scaffold solution-GelMA hydrogel composites may have different moduli from some natural tissues. Therefore, if a tissue with a larger modulus needs to be simulated, the mechanical strength of the composite can be increased by increasing the concentration of GelMA itself.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The composition of the acellular matrix of the present invention is basically close to the natural tissue composition, and can provide good biological functions and simulate the real environment in vivo.

The decellularized scaffold solution-GelMA hydrogel composite material of the present invention has molding conditions and can be directly injected.

The mechanical properties and degradation time of the acellular scaffold solution-GelMA hydrogel composite material of the present invention are controllable, and the gel modulus can be increased and the degradation time can be prolonged by adjusting the content of the acellular matrix or GelMA, so as to be suitable for different applications. tissue repair needs.

Description of drawings

Figure 1 is a flow chart for the preparation of the acellular matrix solution. First, the decellularized scaffold is obtained by decellularization, and the decellularized scaffold is freeze-dried and cut into pieces, hydrolyzed into a solution with pepsin, and finally ultrafiltered to obtain a concentrated matrix fluid.

Figure 2 is the scanning electron microscope images of different proportions of decellularized scaffold solutions and GelMA hydrogels.

Fig. 3 is the actual picture of the decellularized scaffold solution and the decellularized scaffold solution-GelMA hydrogel.

Figure 4 is a diagram of long-term culture of cells in the decellularized scaffold solution-GelMA hydrogel.

Detailed ways

The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be pointed out that the embodiments are only a detailed description of the present invention and should not be regarded as a limitation of the present invention.

The present invention adopts the following steps to prepare the decellularized scaffold solution-GelMA hydrogel composite material.

(1) SD rats were injected with 3% sodium pentobarbital (50 mg/kg) into the abdominal cavity, and their limbs were fixed in a supine position after anesthesia. After sterilization with 75% alcohol, the abdomen was cut with surgical scissors, the abdominal cavity was opened with hemostatic forceps, the intestinal tract was opened with sterile forceps to expose the inferior vena cava and hepatic artery, and a syringe needle was inserted into the portal vein (the pointed end handle into a flat mouth), fix the needle with an arterial clip, and inject heparin saline for anticoagulation. Cut the free arteries and veins, cut the perihepatic ligaments and superior vena cava, transfer the free liver into PBS for washing, and put the liver in a 10 cm plate and store it at -80°C.

(2) The liver frozen at -80°C was repeatedly freeze-thawed 3-4 times and then thawed naturally in a sterile environment at room temperature. Connect the portal vein cannula to the three-way valve and then connect it to the peristaltic pump hose, insert the other end of the hose into the decellularized washing solution, and adjust the flow rate of the peristaltic pump to 20 mL/min. First perfused with sterile PBS for 1 h, followed by 0.1% SDS for 5 h, 1% Triton X-100 for 30 min, 80 U/mL DNase and 5 U/mL RNase for 30 min, and finally with 2% double antibody and 2.5 µg/mL perfusion of amphotericin B in PBS for 2 h. After the decellularization experiment, the decellularized scaffolds were soaked in sterile PBS and stored at -80°C.

(3) Freeze-dry the liver decellularized scaffold obtained in step (2) in a freeze dryer for at least 24 hours. The lyophilized decellularized scaffolds were cut into pieces with sterile surgical scissors, and the cut scaffolds were placed on a constant temperature shaker (37°C, 250 rpm for 6h) and dissolved with pepsin solution (0.5mg pepsin dissolved in 1ml 0.1M HCl) for 12h. - 1 mg of stent dissolved in 1 mL of pepsin solution. Add 1/10 volume of NaCl/10xPBS to the dissolved decellularized scaffold solution to stop the enzymatic reaction, and then adjust the pH to about 7.4 with 5.0M NaOH. Centrifuge at 6,000 rpm with a 3K ultrafiltration tube for 1 h, collect the concentrated decellularized scaffold solution and store it at -20°C.

(4) Prepare 10% wt GelMA hydrogel with distilled water, add 1% wt photoinitiator, and mix with acellular matrix solution in different volume ratios (acellular matrix solution: GelMA=1:9; acellular matrix solution: GelMA =1:4; acellular matrix fluid: GelMA=3:7; acellular matrix fluid: GelMA=2:3; acellular matrix fluid: GelMA=1:1), cured under 365nm UV for 40 s, and scanned under an electron microscope The results obtained under the observation are shown in Figure 1 (a. GelMA only; b. Acellular matrix fluid: GelMA=1:9; c. Acellular matrix fluid: GelMA=1:4; d. Acellular matrix fluid: GelMA =3:7; e. Acellular matrix fluid: GelMA=2:3; f. Acellular matrix fluid: GelMA=1:1), obvious pores and surface acellular matrix can be seen.

The acellular matrix-GelMA gel prepared by the invention is the same as the normal physiological conditions of the human body, and can be directly injected into the body. Scanning electron microscope showed that it had a larger pore structure after molding, which provided conditions for subsequent cell growth and perfect function.

Claims (10)

1. A decellularized scaffold solution-GelMA hydrogel composite material is characterized by comprising the following components: acellular matrix, GelMA and cross-linking agent.

2. A preparation method of a decellularized scaffold solution-GelMA hydrogel composite material is characterized by comprising the following steps:

(1) taking an animal organ which is separated from the carrier, perfusing SDS and Triton X-100 aqueous solution at normal temperature, and removing cells and nucleic acid substances in tissues to obtain a decellularized scaffold;

(2) freeze-drying the acellular scaffold obtained in the step (1), shearing to obtain an acellular matrix block, and digesting the matrix block with pepsin solution for 12 ~ 48 h;

(3) terminating digestion with 10x PBS, and adding NaOH solution to adjust the pH of the acellular matrix solution to be neutral;

(4) carrying out ultracentrifugation on the neutral acellular matrix solution by using an ultrafiltration tube to obtain concentrated acellular matrix solution;

(5) mixing the concentrated acellular matrix solution and GelMA with the mass ratio of 10% according to a certain proportion, ultrasonically stirring the mixture until the mixture is uniformly distributed, adding a corresponding photoinitiator, and carrying out photocrosslinking under the irradiation of ultraviolet light to obtain the acellular scaffold solution-GelMA hydrogel composite material.

3. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method according to claim 2, wherein the preparation method of the acellular scaffold in the step (1) comprises the following steps:

(1) taking fresh and intact animal organs, inserting the fresh and intact animal organs into vein or artery, fixing the insertion tube with vascular by hemostatic forceps, carefully taking out the intact organ, placing in a sterile plate, flushing redundant blood cells with PBS, placing in a refrigerator at-80 deg.C, and repeatedly freezing and thawing for 2 ~ 5 times;

(2) and (3) performing decellularization treatment on the organ, namely perfusing PBS 1 ~ 2h through a vessel for removing blood cells, perfusing 0.1% SDS6 ~ 10 h and Triton X-1000.5 ~ 1h, irrigating the organ with a mixed solution of DNase and RNase for 10 ~ 30min to remove redundant nucleoplasm, washing with PBS for 2 ~ 4h, and storing in a mixed solution containing double antibody and amphotericin.

4. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method according to claim 2, wherein in the step (1), the step of preparing the acellular matrix solution comprises:

(1) freeze drying the acellular scaffold, cutting into powder, digesting the cut matrix with hydrochloric acid solution of pepsin, and treating the treated matrix in a shaking table for 12 ~ 48h until the matrix is dissolved into uniform solution;

(2) adding 1/10 ~ 1/20 volume of 10x PBS solution to the acellular matrix solution to stop enzyme digestion, and adding NaOH (5M) to make the acellular matrix solution neutral (pH =7.0 ~ 7.4.4);

(3) transferring the neutralized acellular matrix solution to an ultrafiltration tube, 5000 ~ 6000gCentrifuging at 4 deg.C for 1 ~ 2h, and collecting concentrated acellular matrix solution.

5. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method according to claim 2, wherein the acellular flow rate in the step (1) is 10 mL/min ~ 20 mL/min.

6. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method thereof according to claim 2, wherein the pepsin solution in the step (2) is a hydrochloric acid solution containing pepsin, wherein the concentration of the hydrochloric acid solution is 0.1 ~ 0.5.5M, and the concentration of the pepsin is 0.5 ~ 5 mg/mL.

7. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method thereof according to claim 6, wherein the ratio of the amount of pepsin to the acellular matrix is 1 ~ 5mg of acellular scaffold dissolved in 1 ~ 10 mL of pepsin hydrochloric acid solution.

8. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method according to claim 1, wherein the concentration of the photoinitiator in the step (5) is 1 ~ 2 wt%.

9. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method thereof according to claim 1, wherein the ultraviolet wavelength in the step (5) is 365nm, and the illumination time is 30 ~ 60 seconds.

10. The acellular scaffold solution-GelMA hydrogel composite material and the preparation method thereof according to claim 1, wherein the mixing ratio of the acellular matrix solution to GelMA in the step (5) is greater than 1:1, wherein the GelMA preparation method is that 0.1g GelMA is dissolved in 1mL aqueous solution or culture medium solution.

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